Human myofibroblastic hepatic stellate cells express Ca-activated K channels that modulate the effects of endothelin-1 and nitric oxide

Background/Aims: High-conductance Ca²⁺-activated K⁺ (BK_Ca) channels modulate the effects of vasoactive factors in contractile cells. It is unknown whether hepatic stellate cells (HSCs) contain BK_Ca channels and what their role in the regulation of HSCs contractility is.Methods: The presence of BK_Ca channels in HSCs was assessed by the patch-clamp technique. The functional role of BK_Ca channels was investigated by measuring intracellular calcium concentration ([Ca²⁺]_i) and cell contraction in individual cells after stimulation with endothelin-1 in the presence or absence of specific modulators of BK_Ca channels.Results: BK_Ca channels were detected by patch-clamp in most of the activated HSCs studied. Incubation of cells with iberiotoxin, a BK_Ca channel blocker, increased both the sustained phase of [Ca²⁺]_i elicited by endothelin-1 and the number of cells undergoing contraction, while the use of NS1619, a BK_Ca channel opener, induced opposite effects. Stimulation of HSCs with S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO)-donor, increased the opening of BK_Ca channels and reduced the effects of endothelin-1. Conversely, iberiotoxin abolished the inhibitory effect of SNAP on endothelin-induced [Ca²⁺]_i increase and cell contraction.Conclusions: Activated human HSCs contain BK_Ca channels that modulate the contractile effect of endothelin-1 and mediate the inhibitory action of NO.     

Excitation-contraction coupling in gastric muscles

Several mechanisms contribute to the regulation of force generated by gastric muscles. Phasic contractions in the stomach are triggered by the propagation of electrical slow waves. These events are associated with an influx of Ca²⁺ and an increase in intracellular Ca²⁺ sufficient to elicit contraction. Entry of Ca²⁺ may be supplemented by the release of Ca²⁺ from intracellular stores. Excitatory agonists enhance the amplitude of slow waves, increase the amplitude of Ca²⁺ transients, and increase the force of phasic contractions. Inhibitory agonists have opposite effects. Excitatory agonists may also enhance release of Ca²⁺ from stores via the production of IP₃. Excitatory and inhibitory agonists may also regulate the sensitivity of the contractile apparatus for Ca²⁺ and therefore alter the contractile response to a given change in intracellular Ca²⁺.     

A mathematical model of vasoreactivity in rat mesenteric arterioles: I. Myoendothelial communication

To study the effect of myoendothelial communication on vascular reactivity, we integrated detailed mathematical models of Ca²⁺ dynamics and membrane electrophysiology in arteriolar smooth muscle (SMC) and endothelial (EC) cells. Cells are coupled through the exchange of Ca²⁺, Cl^?, K⁺, and Na⁺ ions, inositol 1,4,5‐triphosphate (IP₃), and the paracrine diffusion of nitric oxide (NO). EC stimulation reduces intracellular Ca²⁺ ([Ca²⁺ in the SMC by transmitting a hyperpolarizing current carried primarily by K⁺. The NO‐independent endothelium‐derived hyperpolarization was abolished in a synergistic‐like manner by inhibition of EC SK_Ca and IK_Ca channels. During NE stimulation, IP₃diffusing from the SMC induces EC Ca²⁺ release, which, in turn, moderates SMC depolarization and [Ca²⁺]_i elevation. On the contrary, SMC [Ca²⁺]_i was not affected by EC‐derived IP₃. Myoendothelial Ca²⁺ fluxes had no effect in either cell. The EC exerts a stabilizing effect on calcium‐induced calcium release‐dependent SMC Ca²⁺ oscillations by increasing the norepinephrine concentration window for oscillations. We conclude that a model based on independent data for subcellular components can capture major features of the integrated vessel behavior. This study provides a tissue‐specific approach for analyzing complex signaling mechanisms in the vasculature.     

Redox Control of Ion Channel Activity in Vascular Endothelial Cells by Glutathione

Oxidized glutathione (GSSG) is endogenously formed within vascular endothelial cells. The bioactivity of GSSG results in the oxidation of protein thiol groups, leading to changes in protein structure-function relationships. When ion channel protein thiols are the target of oxidation by GSSG, important changes in channel conductance, activity, and gating occur. In this review, we focus on two endothelial cell ion channels, the activities of which influence vascular cell signaling and the nitric oxide signaling pathway. The first channel is the GSSG-operated cation channel that depolarizes the endothelial cell, leading to inhibition of capacitative Ca²⁺ entry. The second channel is the inositol 1,4,5-triphosphate (IP₃)-operated Ca²⁺ channel that is responsible for the agonist-stimulated release of Ca²⁺ from IP₃-sensitive endoplasmic reticulum. GSSG acts to deplete IP₃-sensitive Ca²⁺ stores, thereby attenuating the intracellular Ca²⁺ response to agonist stimulation. Together, these effects indicate that glutathione, which is formed endogenously within the cell, is a key physiological modulator of endothelial cell signaling.     

Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca²⁺. This excessive Ca²⁺ liberation induces Ca²⁺-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca²⁺ release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca²⁺ concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca²⁺ release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca²⁺ release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP₃Rs) of types 2 and 3. POAEE evoked very little Ca²⁺ release and trypsin activation when IP₃Rs of both types 2 and 3 were knocked out. Antibodies against IP₃Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca²⁺ release and trypsin activation. We conclude that Ca²⁺ release through IP₃Rs of types 2 and 3 in the acid granular Ca²⁺ store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.     

Hepatocyte growth factor disrupts cell contact and stimulates an increase in type 3 inositol triphosphate receptor expression, intracellular calcium levels, and apoptosis of rat ovarian surface epithelial cells

The present studies revealed that hepatocyte growth factor (HGF) disrupts cell contact, increases both type 3 IP₃ receptor and intracellular calcium ([Ca²⁺) levels and induces apoptosis of rat ovarian surface epithelial cells (ROSE-179 cells). Type 3 IP₃ receptor was only increased in cells that lost cell contact. Disrupting cell contact by depleting extracellular calcium (Ca²⁺) also resulted in an increase in [Ca²⁺]_i levels and an increase in apoptosis. These responses were prevented by the addition of 0.7 mM Ca²⁺. Actinomycin D and cyclo-heximide prevented apoptosis that resulted from Ca²⁺ removal. In situ hybridization studies revealed that type 3 IP₃ receptor was expressed at relatively low levels by ROSE-179 cells cultured with Ca²⁺ but at high levels in the absence of Ca²⁺. ROSE-179 cells cultured in Ca²⁺-free medium with type 3 IP₃ receptor antisense oligonucleotide lost cell contact but did not show an increase in either type 3 IP₃ receptor protein, [Ca²⁺]_i, or apoptosis. The nonsense oligonucleotide did not alter these responses to Ca²⁺ removal. Thus, the disruption of cell contact by either HGF or Ca²⁺ depletion increases the expression of type 3 IP₃ receptor, which causes an increase in [Ca²⁺]_i and the apoptotic death of ROSE-179 cells.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Insulin receptor signaling for the proliferation of pancreatic β-cells: Involvement of Ca2+ second messengers,IP3, NAADP and cADPR

Insulin has an autocrine/paracrine role through insulin receptors in pancreatic β-cells. Herein, we show the insulin receptor signaling pathway underlying CD38/ADPR-cyclase activation for NAADP/cADPR formation to induce Ca2+ rise, ultimately resulting in β-cell proliferation. Binding of insulin on insulin receptors leads to the activation of IRS/Akt/PI3K/PLC. Activation of PLC generates IP3 and DAG; the former induces Ca²⁺ release, resulting in activation of CD38/ADPR-cyclase for cADPR production via cGMP-dependent mechanism and the latter activates PKC, resulting in activation of ADPR-cyclase for NAADP synthesis. The NAADP-induced Ca²⁺ signal is required for IP₃-induced Ca²⁺ release from the ER. CD38 plays an important role in insulin receptor signaling in β-cells by reflecting a declined sustained Ca²⁺ signal, cADPR levels, and β-cell proliferation in response to insulin in CD38^-/- islets. However, evidence indicates that a hitherto-unidentified ADPR cyclase in addition to CD38 participates in insulin-induced signaling through cADPR and NAADP synthesis. In conclusion, insulin receptor signaling in β-cells employs three Ca²⁺ signaling messengers, IP₃, NAADP, and cADPR through a complex but concerted action of signaling molecules for Ca2+ signaling, which is involved in the proliferation of the islets.     

Acidocalcisomes of Trypanosoma brucei have an inositol 1,4,5-trisphosphate receptor that is required for growth and infectivity

Acidocalcisomes are acidic calcium stores rich in polyphosphate and found in a diverse range of organisms. The mechanism of Ca²⁺ release from these organelles was unknown. Here we present evidence that Trypanosoma brucei acidocalcisomes possess an inositol 1,4,5-trisphosphate receptor (TbIP₃R) for Ca²⁺ release. Localization studies in cell lines expressing TbIP₃R in its endogenous locus fused to an epitope tag revealed its partial colocalization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes. IP₃ was able to stimulate Ca²⁺ release from a chicken B-lymphocyte cell line in which the genes for all three vertebrate IP₃Rs have been stably ablated (DT40-3KO) and that were stably expressing TbIP₃R, providing evidence of its function. IP₃ was also able to release Ca²⁺ from permeabilized trypanosomes or isolated acidocalcisomes and photolytic release of IP₃ in intact trypanosomes loaded with Fluo-4 elicited a transient Ca²⁺ increase in their cytosol. Ablation of TbIP₃R by RNA interference caused a significant reduction of IP₃-mediated Ca²⁺ release in trypanosomes and resulted in defects in growth in culture and infectivity in mice. Taken together, the data provide evidence of the presence of a functional IP₃R as a Ca²⁺ release channel in acidocalcisomes of trypanosomes and suggest that a Ca²⁺ signaling pathway that involves acidocalcisomes is required for growth and establishment of infection.Intracellular Ca²⁺ serves as a second messenger for a variety of cell functions, including secretion, contraction, cell division, and differentiation (1). Cells use two sources of Ca²⁺ for generating signals: Ca²⁺ release from intracellular stores and Ca²⁺ entry across the plasma membrane. Ca²⁺ release from intracellular stores of mammalian cells is controlled by at least three groups of channels: ryanodine receptors, located in the endoplasmic reticulum (ER) and stimulated by cyclic ADP ribose (cADPR) (2); inositol 1,4,5-trisphosphate receptors (IP₃R), also mainly located in the ER and stimulated by IP₃ (3); and two pore channels, preferentially located in acidic calcium stores and stimulated by NAADP (4, 5). IP₃ is generated by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) catalyzed by phosphoinositide-specific phospholipases C (PI-PLCs) (6), which also generate diacylglyerol, whereas ribosyl cyclases catalyze the generation of both cyclic ADP ribose and NAADP from NAD (7). Orthologs to genes encoding some of these enzymes and channels are present in protists (8, 9), suggesting an early appearance of Ca²⁺ signaling during evolution.Cytosolic Ca²⁺ in trypanosomatids such as Trypanosoma cruzi, the etiologic agent of Chagas disease, and Trypanosoma brucei, which belongs to the group of parasites that causes African trypanosomiasis or sleeping sickness, is maintained through the concerted operation of distinct Ca²⁺ transporting systems located in the plasma membrane, ER, mitochondria, and the acidic calcium stores known as acidocalcisomes (10). The IP₃/diacylglycerol pathway was described in T. cruzi more than 20 y ago (11), whereas IP₃ was also detected in T. brucei (12). T. cruzi PI-PLC has been studied in more detail and was shown to be rather unusual in that it does not have a pleckstrin homology domain to bind to the plasma membrane, has a highly charged region between the catalytic X and Y domains, and is N-myristolyated and palmitoylated (13). This lipid modification is important for plasma membrane localization and for stimulation of differentiation of the infective trypomastigotes into amastigotes (14, 15). A gene ortholog to that encoding TcPI-PLC has been found in T. brucei, and the protein product appears to have similar domains to those of the T. cruzi enzyme. Despite the presence of a PI-PLC and the detection of IP₃ in both T. cruzi and T. brucei, early attempts to detect Ca²⁺ release by IP₃ in permeabilized cells were unsuccessful (12, 16).With the completion of several genome projects, it was possible to use bioinformatic methods to identify genes encoding putative IP₃ receptors (8, 17–19) and other channels (9) in protists. Interestingly, apart from one study that described a functional IP₃ receptor in Paramecium tetraurelia (18), biochemical evidence that the other genes identified encode for functional IP₃ receptors is still lacking. Proteomic data from enriched contractile vacuole fractions of T. cruzi (20) included spectra from the putative IP₃Rs, suggesting that these proteins are expressed in subcellular fractions (20).In this work, we report the characterization of the IP₃R from T. brucei, which localizes to their acidocalcisomes. IP₃ was able to release Ca²⁺ from permeabilized cells and isolated acidocalcisomes, and UV light photolysis of caged IP₃ produced a significant increase in intracellular Ca²⁺ in live trypanosomes. Knockdown of the TbIP₃R gene by RNA interference (RNAi) in bloodstream form (BSF) trypanosomes led to growth defects and reduced infectivity in vivo underscoring the relevance of this channel in the parasite.     

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) inducing Ca²⁺ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca²⁺ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca²⁺ release, leading to a significant increase in the activity of Ca²⁺ sparks and contraction in airway SMCs. The increased Ca²⁺ sparks are independent of VDCCs and the associated extracellular Ca²⁺ influx. This format of local Ca²⁺ release results from a direct activation of G protein-coupled, M₃ muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP₃), inducing initial Ca²⁺ release through IP₃ receptors and then further Ca²⁺ release via RyR2 due to a local Ca²⁺-induced Ca²⁺ release process. These findings demonstrate an important mechanism for Ca²⁺ signaling and attendant physiological function in SMCs.     

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear

Extracellular ATP controls various signaling systems including propagation of intercellular Ca²⁺ signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP₃-generating agonist and evokes Ca²⁺ responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP₃ elicited Ca²⁺ responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca²⁺ signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WT cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Px1), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca²⁺ responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca²⁺ responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca²⁺ signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca²⁺-mobilizing second messengers across coupled cells.     

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B₂ bradykinin and M₁ muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP₃ receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP₃ to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca²⁺]_i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca²⁺]_i were blocked by depletion of Ca²⁺ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca²⁺ from IP₃-sensitive Ca²⁺ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.     

T-Type Ca2+ Channels and Pharmacological Blockade: Potential Pathophysiological Relevance

Low-voltage–activated T-type Ca²⁺ channelsare present in most excitable tissues including the heart (mainly pacemakercells), smooth muscle, central and peripheral nervous systems, and endocrinetissues, but also in non-excitable cells, such as osteoblasts, fibroblasts,glial cells, etc. Although they comprise a slightly heterogeneouspopulation, these channels share many defining characteristics: smallconductance (<10 pS), similar Ca²⁺ andBa²⁺ permeabilities, slow deactivation, and avoltage-dependent inactivation rate. In addition, activation at lowvoltages, rapid inactivation, and blockade by Ni²⁺ areclassical properties of T-type Ca²⁺ channels, which areless specific. T-type Ca²⁺ channels are weakly blocked bystandard Ca²⁺ antagonists. Pharmacological blockers arescarce and often lack specificity and/or potency. The physiologicalmodulation of T-type Ca²⁺ currents is complex: they areenhanced by endothelin-1, angiotensin II (AT₁-receptor), ATP,and isoproterenol (cAMP-independent), but are reduced by angiotensin II(AT₂-receptor), somatostatin and atrial natriuretic peptide.Norepinephrine enhances these currents in some cells but decreases them inothers. T-type Ca²⁺ currents have many known or suggestedphysiological and pathophysiological roles in growth (protein synthesis,cell differentiation, and proliferation), neuronal firing regulation, someaspects of genetic hypertension, cardiac hypertrophy, cardiac fibrosis,cardiac rhythm (normal and abnormal), and atherosclerosis. Mibefradil is anew Ca²⁺ antagonist that is effective in hypertension andangina pectoris. Its favorable pharmacological profile and limited sideeffects appear to be related to selective block of T-typeCa²⁺ channels: mibefradil reduces vascular resistance andheart rate without negative inotropy or neurohormonal stimulation, and italso has significant antiproliferative actions.     

The H2O2-Generating Enzyme,Xanthine Oxidase,Decreases Luminal Ca2+ Content of the IP3-Sensitive Ca2+ Store in Vascular Endothelial Cells

Objective: Xanthine oxidase inhibits agonist-stimulated Ca²⁺ signaling in calf pulmonary artery endothelial cells by an H₂O₂-dependent mechanism. We investigated the effect of xanthine oxidase on luminal Ca²⁺ content of the inositol-1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store. Methods: Luminal Ca²⁺ content was estimated from the net release of Ca²⁺ activated by 2,5-di-t-butylhydroquinone (BHQ), an inhibitor of microsomal Ca²⁺ pumps. Results: Initially, xanthine oxidase depleted the IP₃-sensitive Ca²⁺ store of releasable Ca²⁺, but with more prolonged incubation, the enzyme also depleted non-IP₃-sensitive stores. In addition, xanthine oxidase inhibited capacitative Ca²⁺ influx. Similar results were observed when thapsigargin was substituted for BHQ. Conclusions: Depletion of luminal Ca²⁺ content within the IP₃-sensitive Ca²⁺ store contributes to xanthine oxidase inhibition of Ca²⁺ signaling in vascular endothelial cells.     

IP(3)R-mediated Ca(2+) release is modulated by anandamide in isolated cardiac nuclei

Cannabinoids (CBs) are known to alter coronary vascular tone and cardiac performance. They also exhibit cardioprotective properties, particularly in their ability to limit the damage produced by ischaemia reperfusion injury. The mechanisms underlying these effects are unknown. Here we investigate the intracellular localisation of CB receptors in the heart and examine whether they may modulate localised nuclear Ca²⁺ release. In isolated cardiac nuclear preparations, expression of both the inositol 1,4,5-trisphosphate receptor type 2 (IP₃R) and CB receptors (CB₁R and CB₂R) was demonstrated by immunoblotting. Both receptors localised to the nucleus and purity of the nuclear preparations was confirmed by co-expression of the nuclear marker protein nucleolin but absence of cytoplasmic actin. To measure effects of IP₃R and CBR agonists on nuclear Ca²⁺ release, isolated nuclei were loaded with Fluo5N-AM. This dye accumulates in the nuclear envelope. Isolated nuclei responded to IP₃ with rapid and transient Ca²⁺ release from the nuclear envelope. Anandamide inhibited this IP₃-mediated release. Preincubation of nuclear preparations with either the CB₁R antagonist (AM251) or the CB₂R antagonist (AM630) reversed anandamide-mediated inhibition to 80% and 60% of control values respectively. When nuclei were pre-treated with both CBR antagonists, anandamide-mediated inhibition of IP₃-induced Ca²⁺ release was completely reversed. These results are the first to demonstrate the existence of cardiac nuclear CB receptors. They are also the first to show that anandamide can negatively modulate IP₃-mediated nuclear Ca²⁺ release. As such, this provides evidence for a novel key mechanism underlying the action of CBs and CBRs in the heart.     

A model of cytosolic calcium regulation and autacoids production in vascular endothelial cell

Summary A model of vascular endothelial cell is proposed to describe the mechanisms by which cytosolic calcium (Ca_i) is modulated and endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI₂) are released when the cell is stimulated by agonist. The intracellular Ca²⁺ store of the model cell is comprised of a superficial (sc) and a deep (dc) compartment. The dc Ca²⁺ content is refilled by the sc whose [Ca²⁺] is the same as extracellular Ca²⁺. Inositol (1,4,5)-trisphosphate (IP₃) produced by agonist modifies the dc permeability which discharges its Ca²⁺ to the cytosol. The increase of Ca_i induces Ca²⁺ released from the sc. Ca²⁺-activated K⁺ current hyperpolarises the cell. The raised Ca_i releases PGI₂ in the presence of IP₃ while EDRF is released by Ca_i. The model explains satisfactorily the Ca²⁺ transient and autacoids production of the aortie endothelial cell without the need of calcium influx from extracellular space. The cytoplasmic Ca²⁺ oscillations observed in human endothelial cell from umbilical veins were reproduced by the model. Production of EDRF by the artery due to increase in pressre also simulated.     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.