Activators of potassium channels enhance calcium influx into endothelial cells as a consequence of potassium currents

Summary Ca²⁺ influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca²⁺ influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K⁺ channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca²⁺]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 mol/l) and cromakalim (1 mol/l) elicited outward currents carried by K⁺ and significantly prolonged the Ca²⁺-dependent K⁺ currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca²⁺ indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1–1 mol/l elicited long-lasting increases in [Ca²⁺)_i from 100 ± 10 to 550 ± 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K⁺. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca²⁺]_i when applied during the decline of [Ca²⁺]_i after the initial peak. Cicletanine elicited K⁺ currents in resting and attenuated K⁺ currents in bradykinin-stimulated cells. It elevated [Ca²⁺]_i even in the absence of extracellular Ca²⁺ and in K⁺-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K⁺ channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca²⁺]_i secondary to their activation of K⁺ channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca²⁺ influx. In this way, the two drugs may promote Ca²⁺-dependent formation of endothelium-derived relaxing factor. Send offprint requests to A. Lückhoff at the above address     

Histamine H1-receptors in HL-60 monocytes are coupled to Gi-proteins and pertussis toxin-insensitive G-proteins and mediate activation of Ca2+ influx without concomitant Ca2+ mobilization from intracellular stores

The results of binding studies suggest the presence of histamine H₁-receptors in human monocytes, but it is not known whether these receptors are functionally active. This prompted us to study the effects of histamine (HA) on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and superoxide anion (O₂ ^– ) formation in HL-60 cells differentiated towards monocytes with 1,25-dihydroxychole-calciferol. In HL-60 monocytes, HA increased [Ca²⁺]_i with a half-maximal effect at 8 M and a maximum at 30–100 M. Pertussis toxin (PTX) partially inhibited the stimulatory effects of HA on [Ca²⁺]_i. Betahistine, a weak partial H₁-receptor agonist, also increased [Ca²⁺]_i, whereas H₂- and H₃-receptor agonists were ineffective. H₁- but not H₂- and H₃-receptor antagonists inhibited HA induced rises in [Ca²⁺]_i. HA-induced rises in [Ca²⁺]_i were desensitized in a homologous manner and were also inhibited by the activator of protein kinase C, 4\-phorbol 12-myristate 13-acetate. Various protein kinase C inhibitors did not interfere with homologous desensitization. The stimulatory effects of HA on [Ca²⁺]_i were completely dependent on the presence of extracellular Ca²⁺ and were inhibited by the blocker of non-selective cation (NSC) channels, 1-{\-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl}-1 H-imidazole hydrochloride (SK & F 96365). HA was much less effective than the chemotactic peptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), to induce rises in [Ca²⁺]_i. Unlike fMLP, HA did not activate O₂ ^– formation. Our data indicate that HL-60 monocytes possess H₁-receptors coupled to heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) of the G_i-family and PTX-insensitive G-proteins which mediate activation of NSC channels without concomitant activation of Ca²⁺ mobilization from intracellular stores, that homologous desensitization of HA-induced Ca²⁺ influx is independent of protein kinase C and that the stimulatory effect of HA on Ca²⁺ influx is too small to result in activation of O_{2 –} formation. Correspondence to: R. Seifert at the above address     

Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

Summary The effects of ₁-adrenoceptor stimulation by phenylephrine (PE) and -adrenoceptor stimulation by isoprenaline (ISO) on Ca²⁺ current (I_Ca) and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of I_Ca, whereas large increases of peak I_Ca were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca²⁺]_i and, initially, an increase in the height of peak [Ca²⁺]_i transients. When the diastolic [Ca²⁺]_i was increased to a greater extent, the amplitude of [Ca²⁺]_i transients was decreased. Simultaneous measurements of [Ca²⁺]_i and membrane potential showed that the increase in diastolic [Ca²⁺]_i was associated with a depolarization of the membrane, and the greater amplitude of [Ca²⁺]_i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca²⁺]_i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca²⁺]_i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca²⁺]_i transients with only minor changes in diastolic [Ca²⁺]_i. These results suggest that PE and ISO increase the amplitude of [Ca²⁺]_i transients in rat atrium in different ways. The increase in [Ca²⁺]_i transients in response to -adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca²⁺ channels causing a greater Ca²⁺ influx and a release of more Ca²⁺ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca²⁺]_i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na⁺-Ca²⁺ exchange mechanism. The increase in the amplitude of the [Ca²⁺]_i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca²⁺]_i and the prolongation of the AP. Send offprint requests to H. Nawrath at the above address     

Identification of a B2-bradykinin receptor linked to phospholipase C and inhibition of dopamine stimulated cyclic AMP accumulation in the human astrocytoma cell line D384

Summary We have examined the activation of a phospholipase C signal transduction pathway by a B₂-bradykinin receptor in the human astrocytoma cell line D384 and how this influences D1-dopamine receptor stimulated cyclic AMP accumulation. Addition of bradykinin to D384 cells resulted in a concentration-dependent (10^–11 – 10^–6 M) increase in the accumulation of [³H]inositol phosphates and a similar concentration-dependent transient increase in specific [³H]-phorbol-12,13-dibutyrate binding which is indicative of translocation of protein kinase C from the cytosol to the membrane. Changes in intracellular Ca²⁺ of single cells, measured using the fluorescent indicator dye fura-2, indicated that bradykinin produced a rapid, but transient, increase in intracellular calcium. The Ca²⁺ response was largely independent of extracellular Ca²⁺ supporting the idea that receptor activation leads to mobilization of Ca²⁺ from intracellular stores. However, extracellular Ca²⁺ was required for a response to a rechallenge with bradykinin. The bradykinin B₂-receptor agonist kallidin increased cytosolic Ca ²⁺ in a similar manner to bradykinin. The Ca²⁺ response to bradykinin could be partially reduced in the presence of the B₂-receptor antagonist [d-Arg¹⁰-Hyp,d-Phe⁷,-(2-Thienyl)-Ala⁵,⁸]-bradykinin, whereas the B₁-receptor agonists (Des-Arg⁹]-bradykinin and [Des-Arg¹⁰]-kallidin were ineffective. Bradykinin was also found to attenuate dopamine stimulated cyclic AMP accumulation in D384 cells, at similar concentrations previously observed to stimulate the phospholipase C signal transduction pathway, in the presence of the phosphodiesterase inhibitor, rolipram. In contrast, no attenuation was observed in the presence of the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine, although the level of dopamine stimulated cyclic AMP observed was lower than in the presence of rolipram. Furthermore, the effect of bradykinin could be mimicked by a calcium ionophore, but not a phorbol ester. These data suggest that D384 cells express a B₂ bradykinin receptor coupled to polyphosphoinositide-specific phospholipase C. Activation of this receptor results in elevated levels of cytosolic Ca²⁺ and to a reduction of D1-dopamine receptor stimulated cyclic AMP accumulation. Correspondence to A. J. Balmforth at the above address     

Glutamate in Life and Death of Retinal Amacrine Cells*

• 1.Glutamate is the neurotransmitter released by bipolar cells at their synapses with amacrine cells. The amacrine cells express ionotropic (NMDA, AMPA and kainate) and metabotropic (mGluR1, mGluR2, mGluR4 and mGluR7) glutamate receptors and may take up glutamate from the synaptic cleft.
• 2.Activation of the ionotropic glutamate receptors increases the intracellular free calcium concentration ([Ca²⁺]_i), owing to Ca²⁺ entry through the receptor-associated channels as well as through voltage-gated Ca²⁺ channels. The [Ca²⁺]_i response to glutamate may be amplified by Ca²⁺-induced Ca²⁺ release from intracellular sources.
• 3.Activation of NMDA and non-NMDA glutamate receptors stimulates the release of GABA and acetylcholine from amacrine cells. GABA is released by a Ca²⁺-dependent mechanism and by reversal of the neurotransmitter transporter.
• 4.Excessive activation of glutamate receptors during ischemia leads to amacrine cell death. An increase in [Ca²⁺]_i due to Ca²⁺ influx through NMDA and AMPA/kainate receptor channels is related to cell death in studies in vitro. In other studies, it was shown that nitric oxide may also take part in the process of cell damage during ischemia.

     

Calcium signalling by G protein-coupled sphingolipid receptors in bovine aortic endothelial cells

Besides its role as a putative second messenger releasing Ca²⁺ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), amounting to maximally about 230 nM. Removal of extracellular Ca²⁺ revealed that SPP-induced [Ca²⁺]_i elevations were due to both release of Ca²⁺ from intracellular stores and influx of extracellular Ca²⁺. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca²⁺]_i by 83%, in line with the previously reported involvement of G proteins of the G_i/o family in SPP signalling in other cell types. In contrast to other [Ca²⁺]_i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca²⁺. Furthermore, SPP also did not cause activation of either phospholipase D or A₂. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca²⁺ _i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca²⁺]_i differed by more than two orders of magnitude, with the EC₅₀ values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a G_i/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.     

Effects of P2 Purinoceptor Agonists on Membrane Potential and Intracellular Ca2+ of Human Cardiac Endothelial Cells

Abstract: Vasoactive agonists like adenosine-5′-triphosphate (ATP) increase intracellular Ca²⁺ ([Ca²⁺]_i) in vascular endothelial cells with an initial peak due to inositol 1,4,5-triphosphate-mediated Ca²⁺ release from intracellular stores followed by a sustained plateau that is dependent on the presence of extracellular Ca²⁺, thus leading to an increased synthesis and release of prostacyclin and nitric oxide. We studied the effects of nucleotides on membrane potential and [Ca²⁺]_i in confluent human microvascular cardiac endothelial cells obtained from patients with dilated cardiomyopathy. The whole-cell configuration of the patch-clamp technique and a confocal laser scanning microscope employing fluo-3 as a Ca²⁺ indicator were used. Both uridine-5′-triphosphate (UTP) and 2-methylthioadenosine-5′-triphosphate (2MeSATP) induced depolarizations in human microvascular cardiac endothelial cells and increased [Ca²⁺]_i with a rank order of potency 2MeSATP>ATP=UPP (EC₅₀ values (in μM) were 0.084 2MeSATP, 0.67 ATP and 1.1 UTP). This suggests that both P_2u and P_2y purinoceptors are present on human microvascular cardiac endothelial cells. Maximal [Ca²⁺]_i responses of confluent human microvascular cardiac endothelial cell monolayers to UTP were lower when compared to 2MeSATP. Nucleotide-induced increases in [Ca²⁺]_i consisted of a transient peak, which was also observed in the absence of extracellular Ca²⁺, and a sustained [Ca²⁺]_i plateau. This plateau, which was not observed in all monolayers studied, was not markedly influenced by increasing extracellular [K⁺]. Previous incubation with thapsigargin abolished ATP-induced increases of [Ca²⁺]_i. It is concluded that human microvascular cardiac endothelial cells express both P_2y and P_2u purinoceptors. P₂ purinoceptor agonists release Ca²⁺ from intracellular thapsigargin-sensitive stores and stimulate capacitative Ca²⁺ influx pathways. K⁺ efflux through Ca²⁺-dependent K⁺ (K_ca) channels does not play a major role in the regulation of nucleotide-induced Ca²⁺ influx in human microvascular cardiac endothelial cells, which might be related to an impaired function of the cells.     

ROLE OF CYCLIC NUCLEOTIDES IN THE CONTROL OF CYTOSOLIC Ca2+ LEVELS IN VASCULAR ENDOTHELIAL CELLS

• 1 Endothelial cells have a key role in the cardiovascular system. Most endothelial cell functions depend on changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) to some extent and Ca²⁺ signalling acts to link external stimuli with the synthesis and release of regulatory factors in endothelial cells. The [Ca²⁺]_i is maintained by a well‐balanced Ca²⁺ flux across the endoplasmic reticulum and plasma membrane.
• 2 Cyclic nucleotides, such as cAMP and cGMP, are very important second messengers. The cyclic nucleotides can affect [Ca²⁺]_i directly or indirectly (via the actions of protein kinase (PK) A or PKG‐mediated phosphorylation) by regulating Ca²⁺ mobilization and Ca²⁺ influx. Fine‐tuning of [Ca²⁺]_i is also fundamental to protect endothelial cells against damaged caused by the excessive accumulation of Ca²⁺.
• 3 Therapeutic agents that control cAMP and cGMP levels have been used to treat various cardiovascular diseases.
• 4 The aim of the present review is to discuss: (i) the functions of endothelial cells; (ii) the importance of [Ca²⁺]_i in endothelial cells; (iii) the impact of excessive [Ca²⁺]_i in endothelial cells; and (iv) the balanced control of [Ca²⁺]_i in endothelial cells via involvement of cyclic nucleotides (cAMP and cGMP) and their general effectors.

     

Enhancement by captopril of bradykinin-induced calcium transients in cultured endothelial cells of the bovine aorta

Using microscopic fluorometry and fura-2-loaded cultured bovine aortic endothelial cells, we determined the effects of captopril, an angiotensin converting enzyme (ACE) inhibitor, on bradykinin-induced Ca²⁺ transients in endothelial cells. In the presence of extracellular Ca²⁺, 10⁻⁹ M bradykinin induced an early rise in the transients followed by sustained elevations of cytosolic calcium concentration ([Ca²⁺]_i). Bradykinin concentration-dependently increased [Ca²⁺]_i (EC₅₀ 6.7 × 10⁻⁹ M). Captopril, 10⁻⁵ M, enhanced and prolonged the bradykinin-induced Ca²⁺ transients and shifted the concentration-response curve to the left (EC₅₀ 8.5 × 10⁻¹⁰ M). In porcine coronary aterial strips with intact endothelium, cumulative applications of bradykinin induced an endothelium-dependent relaxation during prostaglandin F_2α-induced contraction (EC₅₀ = 2.0 × 10⁻⁹ M). Treatment with 10⁻⁵ M captopril enhanced the bradykinin-induced relaxation and shifted the concentration-response curve to the left (EC₅₀ = 7.6 × 10⁻¹⁰ M). Thus, captopril enhances the bradykinin-induced relaxation by mechanisms mainly dependent on the endothelium, namely the inhibition of ACE.     

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

Stimulation of cellular free Ca2+ elevation and inhibition of store-operated Ca2+ entry by kazinol B in neutrophils

Kazinol B, a natural isoprenylated flavan, stimulated the [Ca²⁺]_i elevation in the presence or absence of Ca²⁺ in the medium. Treatment with chymotrypsin or phorbol 12-myristate 13-acetate to shedding of l-selectin had no effect on subsequent kazinol B-induced Ca²⁺ response. Upon initial cyclopiazonic acid (CPA) treatment in the absence of external Ca²⁺, the subsequent [Ca²⁺]_i rise followed by challenge with kazinol B was greatly diminished. The ryanodine receptor blockers, 8-bromo-cyclic ADP-ribose and ruthenium red did not affect kazinol B-evoked Ca²⁺ release from internal stores. However, the inhibitors of sphingosine kinase, dimethylsphingosine, but not dihydrosphingosine, inhibited kazinol B-induced Ca²⁺ release. Kazinol B-induced [Ca²⁺]_i rise was not affected by two nitric oxidase inhibitors, N-(3-aminomethyl)benzylacetamidine (1400W) and 7-nitroindazole, cytochalasin B and Na⁺-deprivation. This response was slightly attenuated by 2-aminoethyldiphenyl borate (2-APB), a d-myo-inositol 1,4,5-trisphosphate (IP₃) receptor blocker, and by genistein, a general tyrosine kinase inhibitor. However, the Ca²⁺ response was greatly diminished by two actin filament reorganizers, calyculin A and jasplakinolide, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), an inhibitor of phosphoinositide 3-kinase, N-(3-aminomethyl)benzylacetamidine (SB 203580), the p38 mitogen-activated protein kinase inhibitor, 1-[6-[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, and by 0.3 mM La³⁺ or Ni²⁺. Kazinol B did not evoke any appreciable Ba²⁺ and Sr²⁺ entry into cells. The Ca²⁺ entry blockers, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), but not cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), inhibited a kazinol B-induced [Ca²⁺]_i rise. Kazinol B had no effect on the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity. In a Ca²⁺-free medium, kazinol B inhibited the subsequent Ca²⁺ addition, resulting in robust entry in CPA- and formyl peptide-activated cells. Kazinol B produced a concentration-dependent reduction in the mitochondrial membrane potential. These results indicate that kazinol B stimulates Ca²⁺ release from internal Ca²⁺ store, probably through the sphingosine 1-phosphate and IP₃ signaling, and activates external Ca²⁺ influx mainly through a non-store-operated Ca²⁺ entry (non-SOCE) pathway. Inhibition of SOCE by kazinol B is probably attributable to a break in the Ca²⁺ driven force of mitochondria.     

cAMP- and diacylglycerol-mediated pathways elevate cytosolic free calcium concentration via dihydropyridine-sensitive, ω-conotoxin-insensitive calcium channels in normal rat anterior pituitary cells

Summary The effect of 8-bromocyclic AMP (8-Br-cAMP) and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, on cytosolic free calcium concentration ([Ca²⁺]_i) in normal rat anterior pituitary cells was examined. [Ca²⁺]_i was monitored directly by the fluorescent indicator fura-2. 8-Br-cAMP as well as PMA elevated [Ca²⁺]_i in a concentration-dependent manner. Forskolin (10 mol/l), which activates adenylate cyclase, and 1-oleoyl-2-acetyl-glycerol (10 mol/l), another activator of protein kinase C, also increased [Ca²⁺]_i. Both the 8-Br-cAMP (2 mmol/l)- and the PMA (100 nmol/l)-induced increase in [Ca²⁺]_i was dependent on the presence of extracellular calcium and could be inhibited by the calcium channel blockers Mg²⁺ and nifedipine, but not by onotoxin (100 nmol/l). The half-maximally inhibitory concentrations of Mg²⁺ and nifedipine were about 12 mmol/l and 160 nmol/l, respectively, for the [Ca²⁺]_i response to 8-Br-cAMP (2 mmol/l), and were about 6 mmol/l and 400 nmol/1, respectively, for the PMA (100 nmol/1)-induced increase in [Ca²⁺]_i. The sodium channel blocker tetrodotoxin (5 mol/l) had no influence on the effect of 8-Br-cAMP (2 mmol/l) or PMA (100 nmol/l) on [Ca²⁺]_i. After pretreatment for 3 min with PMA (100 nmol/l), the subsequent K⁺ (100 mmol/l)- or arachidonic acid (3 mol/l)-induced increase in [Ca²⁺]_i was decreased by about 50%. By contrast, pretreatment (3 min) with 8-Br-cAMP (2–10 mmol/1) markedly enhanced the subsequent [Ca²⁺]_i response to K⁺ (100 mmol/l), and left the effect of arachidonic acid (3 mol/l) on [Ca²⁺]_i unimpaired. These data indicate that both cAMP- and diacylglycerol-mediated pathways increase [Ca²⁺ _i in normal rat anterior pituitary cells via an influx of extracellular Ca²⁺ through dihydropyridine-sensitive, -conotoxin-insensitive voltage-dependent calcium channels. These second messengers may thus be involved in Ca²⁺ channel activation by hypothalamic releasing hormones. Effects of cAMP- or diacylglycerol-induced pathways on anterior pituitary function may not be independent of but be mediated also by changes in [Ca²⁺]_i. However, substantial differences appear to exist in how cAMP and diacylglycerol influence voltage-dependent calcium channels.     

Biphasic increase in cytosolic free calcium induced by bradykinin and histamine in cultured tracheal smooth muscle cells: is the sustained phase artifactual?

The effects of bradykinin (BK) and histamine on intracellular Ca²⁺ ([Ca²⁺]i) were studied in fura-2-loaded guinea-pig tracheal smooth muscle cells in culture. BK, at 10 nM, and histamine, at 100 M, induced a rise in [Ca²⁺]i which was inhibited by the B2 antagonist Hoe 140 and by the H1 antagonist triprolidine, respectively. This rise in [Ca²⁺]_i is biphasic, consisting of a rapid transient phase followed by a sustained phase. The transient phase, induced by either BK or histamine, was strongly inhibited by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor, usually used to deplete certain intracellular Ca²⁺-stores. Ni²⁺ (4 mM) did not affect the transient phase but abolished the sustained phase when cells were stimulated by BK, further supporting the fact that the transient phase was only dependent on intracellular Ca²⁺ pools. The sustained phase was partially (for BK) and completely (for histamine) inhibited by 30 M Mn²⁺. This effect could be completely reversed by the addition of DTPA, a cell-impermeant chelator of Mn²⁺, indicating that the Mn²⁺ exerted its effect extracellularly. The presence of 1 mM probenecid (an inhibitor of a membrane organic anion transporter that extrudes fura-2) drastically inhibited the sustained phase by more than 77010 for BK and 88010 for histamine. Our results suggest that the effects of BK and histamine on airway smooth muscle cells are mediated via bradykinin B2 receptors and histamine H1 receptors, respectively whose activation allows the rapid transient rise in [Ca²⁺]i from thapsigargin-sensitive intracellular Ca²⁺ pools. The sustained phase is proposed to be drastically influenced by an acceleration of fura-2 extrusion during the increase of [Ca²⁺]_i via a probenecid-sensitive mechanism.Abbreviations BK bradykinin - [Ca²⁺]_i cytosolic free calcium - CICR Ca²⁺-induced Ca²⁺ release - DMEM Dulbecco's modified Eagle's medium - DTPA diethylenetriaminepentaacetic acid - EGTA ethylene glycol-bis(b-aminoethylether) N,N,N,N-tetraacetic acid - FCS fetal calf serum - HEPES 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethansulfonic acid - IP inositol phosphates - TG thapsigargin - TSMC tracheal smooth muscle cells Correspondence to: C. Bronner     

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca²⁺ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and the increase of [Ca²⁺]_i by OxLDL or by LPC was inhibited by La³⁺ or heparin. LPC failed to increase [Ca²⁺]_i in the presence of an antioxidant tempol. In addition, store-operated Ca²⁺ entry (SOC), which was evoked by intracellular Ca²⁺ store depletion in Ca²⁺-free solution using the sarcoplasmic reticulum Ca²⁺ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca²⁺]_i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La³⁺ or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca²⁺ to 1 µM activated large-conductance Ca²⁺-activated K⁺ (BK_Ca) current spontaneously, and this activated BK_Ca current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca²⁺-permeable Ca²⁺-activated NSC current and BK_Ca current simultaneously, thereby increasing SOC.     

Effects of a myosin light chain kinase inhibitor, wortmannin, on cytoplasmic Ca2+ levels, myosin light chain phosphorylation and force in vascular smooth muscle

Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 2157–2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 M) decreased MLC phosphorylation and the amplitude of contractions induced by high K⁺ (72.7 mM) to a level seen at rest. This occurred without a change in cytosolic Ca²⁺ levels ([Ca²⁺]_i). In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 M) and prostaglandin F₂ (PGF₂, 10 M) without a change in the [Ca²⁺]_i. On the other hand, wortmannin (1 or 10 M) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF₂ to a level seen at rest. In the absence of external Ca²⁺, caffeine (20 mM) induced a transient increase in [Ca²⁺]_i and force with an increase in MLC phosphorylation. Wortmanmn completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [Ca²⁺]_i. In the absence of external Ca²⁺, phenylephrine induced a small transient increase in [Ca²⁺]_i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca²⁺]_i and MLC phosphorylation. Wortmannin (1 M) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca²⁺]_i nor the sustained contraction. Wortmannin inhibited the Ca²⁺-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the Ca²⁺-independent, ATP-induced contraction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entry of Ca²⁺ or through the release of Ca²⁺ from the sarcoplasmic reticulum. The results also suggest that the activation of receptors by norepinephrine and PGF₂. induces a contraction via a MLC phosphorylation-independent pathway or through a pathway which is dependent on the resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in mouse aortic smooth muscle cells

Background and purpose:

Reduction of intracellular calcium ([Ca²⁺]_i) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca²⁺ mobilization during SMC contraction influenced NO efficacy.

Experimental approach:

Isometric contractions by depolarization (high potassium, K⁺) or α-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca²⁺]_i (Fura-2) were measured in aortic segments from C57Bl6 mice.

Key results:

Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K⁺ solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca²⁺]_i, which was suppressed by sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K⁺-stimulated segments did not parallel a decline of [Ca²⁺]_i. However, stimulation (BAY K8644) of L-type Ca²⁺ influx diminished, while inhibition (nifedipine, 1–100 nM) augmented the relaxing capacity of NO.

Conclusions and implications:

In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca²⁺ re-uptake into the SR and was prominent when intracellular Ca²⁺ was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca²⁺]_i, presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca²⁺ influx, and L-type Ca²⁺ channel blockers increase the vasodilator efficacy of NO.     

Palytoxin-induced increase in endothelial Ca2+ concentration in the rabbit aortic valve

Palytoxin (PTX) is one of the most potent toxins isolated from marine coelenterates of the genus Palythoa. It induces depolarization in various types of cells by increasing the permeability for monovalent cations. It has been reported that PTX induces endothelium-dependent relaxation of vascular smooth muscle. In this study, we examined the effect of PTX on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the endothelium of rabbit aortic valves loaded with fluorescent Ca²⁺ indicators, fura-PE3 or fluo-3. PTX (10pM-300nM) irreversibly increased endothelial [Ca²⁺]_i in a concentration-dependent manner. ATP and thapsigargin also increased [Ca²⁺]_i. Imaging of [Ca²⁺]_i with a confocal microscope revealed that PTX increased [Ca²⁺]_i in all endothelial cells studied (n=13). An inorganic Ca²⁺ entry blocker, La³⁺ (30μM), had no effect on the increase in [Ca²⁺]_i induced by PTX whereas it inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. The PTX-induced increase in [Ca²⁺]_i was partially inhibited by ouabain and was abolished by removal of external Ca²⁺ although decrease of Na⁺ concentration in the incubation medium was ineffective. Activation of protein kinase C by 1μM 12-deoxyphorbol 13-isobutyrate or inhibition of phosphatase by 10nM calyculin-A had no effect on the increase in [Ca²⁺]_i induced by PTX, whereas both agents inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. Mn²⁺ influx, measured by the quenching of fura-PE3 fluorescence, was accelerated by ATP or thapsigargin, but not by PTX. These results suggest that PTX increases [Ca²⁺]_i in the endothelium of the rabbit aortic valve by increasing Ca²⁺ influx through a pathway which is different from that activated by ATP or thapsigargin. Received: 28 February 1997     

Potentiation by endothelin-1 of Ca2+ sensitivity of contractile elements depends on Ca2+ influx through L-type Ca2+ channels in the canine cerebral artery

• 1.1. Endothelin-1 (ET-1) contracted canine cerebral artery in a concentration-dependent manner with an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and at higher concentrations it produced a greater contraction with a smaller increase in [Ca²⁺]_i.
• 2.2. Ca²⁺ channel antagonist such as d-cis-diltiazem inhibited the tension more effectively than the [Ca²⁺]_i increased by ET-1.
• 3.3. In Ca²⁺-free solution containing 0.2 mM EGTA, ET-1 elicited a transient increase in [Ca²⁺]_i and tension.
• 4.4. In the Staphylococcus aureus α-toxin-permeabilized artery, ET-1 shifted the pCa-tension relationship leftwards in the presence of GTP.
• 5.5. These findings suggest that ET-I contracts the canine cerebral artery by increasing not only the Ca²⁺ influx through L-type Ca²⁺ channels, but also Ca²⁺ release from the intracellular storage sites, and also Ca²⁺ sensitivity of contractile elements. The degree of Ca²⁺ sensitivity is strongly affected by [Ca²⁺]_i which is increased by the Ca²⁺ influx through L-type Ca²⁺ channels.