Intracellular calcium mobilization on stimulation of the muscarinic cholinergic receptor in chick limb bud cells

Summary Cell suspensions of chick limb buds (stage 23/24) were loaded with the fluorescent Ca²⁺ chelator chlorotetracycline. Fluorescence was monitored in a spectrofluorometer. Stimulation with acetylcholine induced a fluorescence decrease, indicating intracellular Ca²⁺ mobilization. The fluorescence decrease triggered by acetylcholine was inhibited by muscarinic but not by nicotinic antagonists, indicating that a muscarinic acetylcholine receptor is involved. The muscarinic receptor in the chick limb bud has a characteristic pharmacological profile: acetylcholine, carbachol and acetyl--methylcholine functioned as full agonists triggering maximal fluorescence decrease. Bethanechol was less effective, producing only one-third of the maximum response. Pilocarpine and oxotremorine, two classical agonists in other systems, were ineffective and functioned as antagonists. In the chick limb bud, cholinesterase, choline acetyltransferase and the presence of a muscarinic receptor have been demonstrated in previous studies. The present experiments show that stimulation of the embryonic muscarinic receptor leads to intracellular Ca²⁺ mobilization.     

Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

cGMP production is coupled to Ca-dependent nitric oxide generation in rabbit parotid acinar cells

We investigated the mechanism of guanosine 3′,5′-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a β-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca²⁺ mobilization, because intracellular Ca ²⁺ elevating reagents, such as thapsigargin and the Ca²⁺ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca²⁺ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by N^G-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca²⁺. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca²⁺ mobilization.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

Muscarinic acetylcholine responses in the early embryonic chic retina

The action of acetylcholine on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in early embryonic chick retinae. Whole neural retinae were isolated from embryonic day 3 (E3) chicks and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). Increases in [Ca²⁺]_i were evoked by the puff application of acetylcholine at concentration than 0.1 μM. The Ca²⁺ response became larger in dose–dependant manner up to 10 μM of acetylcholine applied. The rise in [Ca²⁺]_i was not due to the influx of Ca⁺² through calcium channels, but to the release of Ca²⁺ from internal stores. A calcium channel antagonist, nifedipine, which completely blocks the Ca²⁺ rise caused by depolarization with 100 mM K⁺, had no effects on the acetylcholine response and the Ca²⁺ response to acetylcholine occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolished the response to 10 μM acetylcholine, whereas d-tubocurarine of 100 μM had no effects. Two muscarinic agonists, muscarine and carbamylcholine (100 μM each), evoked comparable responses with that to 10 μM acetylcholine. The developmental change of the muscarinic response was examined from E3 to E13. The Ca2+ response to 100 μM carbamylcholine was intense at E3-E5, then rapidly declined until E8. The muscarinic Ca²⁺ mobilization we found in the early embryonic chick retina may be regarded as a part of the “embryonic muscarinic system” proposed by Drew's group, which appears transiently and ubiquitously at early embryonic stages in relation to organogenesis. 1994 John Wiley & Sons, Inc.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Activation by calcium of membrane channels for potassium in exocrine gland cells

In the rat parotid salivary gland, fluid secretion is regulated by alterations in fluxes of monovalent ions. , stimulation of muscarinic, α-adrenergic or substance P receptors provokes a biphasic increase in membrane permeability to K⁺ which can be conveniently assayed as efflux of ⁸⁶Rb. The increased ⁸⁶Rb flux is thought to arise in response to a receptor mediated elevation in [Ca²⁺]_i which activates Ca²⁺-activated K⁺-channels. The biphasic nature of the response is presumably due to a biphasic mode of Ca²⁺ mobilization by secretagogues; a transient response reflects release of a finite pool of Ca from an intracellular store while a more sustained phase results from Ca entry through receptor operated Ca channels or gates. Calcium also mediates an increased Na⁺ entry which in turn activates the Na⁺, K⁺-pump. The mechanism involved in the regulation of monovalent ion channels by Ca²⁺ is not understood.     

Effects of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, on platelet activity

In the present study, the mechanism of antiplatelet activity of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, was investigated. DK-002 inhibited the thrombin, collagen, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC₅₀ values of 120, 27, and 47 μM, respectively. DK-002 also inhibited thrombin-induced dense granule secretion, thromboxane A₂ synthesis, and [Ca²⁺]_i elevation in platelets. DK-002 did not show any significant effect on ADP-induced inhibition of cyclic AMP elevation by prostaglandin E₁, but DK-002 was confirmed to inhibit ADP-induced [Ca²⁺]_i elevation and shape change. DK-002 inhibited 4-bromo-A23187-induced [Ca²⁺]_i elevation in the presence of creatine phosphate/creatine phosphokinase (CP/CPK, a ADP scavenging system) and indomethacin (a specific inhibitor of cyclooxygenase). DK-002 also inhibited Ca²⁺ mobilization in thrombin- or 4-bromo-A23187-stimulated platelets through its inhibitory effects on both Ca²⁺ release from intracellular stores and Ca²⁺ influx, in the presence of CP/CPK and indomethacin. Taken together, the present study shows that DK-002 has inhibitory effects on stimulation of platelets, and suggests that its antiplatelet activity might be related to the inhibitory mechanism on Ca²⁺ mobilization in stimulated platelets.     

Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit

G protein–coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca²⁺, and phosphatidylinositol 4,5-bisphosphate (PIP₂), to modulate N-type voltage-gated Ca²⁺ (Ca_V2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which G_qPCRs inhibit Ca_V2.2 channel current are not completely understood. Here, we report that the location of Ca_V β subunits is key to determining the voltage dependence of Ca_V2.2 channel modulation by G_qPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M₁ receptors, together with Ca_V N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for Ca_V2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of Ca_V2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of Ca_V2.2 current after M₁ receptor activation was markedly reduced and delayed, whereas the delay between PIP₂ hydrolysis and inhibition of Ca_V2.2 current was decreased. When the Gβγ-insensitive Ca_V2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M₁ receptors act through Gβγ to inhibit Ca_V2.2 channels bearing membrane-localized Ca_V β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M₁ muscarinic inhibition of Ca_V2.2 channels, whereas it increased inhibition mediated by PIP₂ depletion. Together, our results indicate that, with membrane-localized Ca_V β subunits, Ca_V2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP₂-mediated voltage-independent regulation. Thus, the voltage dependence of G_qPCR regulation of calcium channels can be determined by the location of isotype-specific Ca_V β subunits.     

In vivo adaptative regulation of muscarinic receptors and muscarinic stimulation-induced Ca2+ mobilization during short-term heat exposure in rat parotid glands

1. Adaptation of muscarinic receptors (MR)—muscarinic stimulation—induced intracellular Ca²⁺ mobilization during short-heat exposure (33°C).2. Heat-exposure for 48 hr decreased the carbachol (CCh)-stimulated cytosolic C²⁺ concentration increase.3. The number of MR on cell surface increased transiently at 24 hr with a subsequent decrease at 48 hr.4. CCh-stimulated inositol trisphosphate (IP₃) formation decreased at 48 hr.5. In saponin-permeabilized cells, 1,4,5-IP₃-induced ⁴⁵Ca²⁺ release decreased at 24 hr.6. The data suggest that the adaptation for increased muscarinic stimulation occurs at IP₃ generating sites as well as at intracellular IP₃ receptor sites during heat exposure.     

Ca2+-mobilizing receptors of gastrulating chick embryo

1. Gastrulating chick embryo cells (stages 3–5 by HH) possess Ca²⁺-mobilizing receptors for ACh and ATP; insulin and noradrenaline have a weaker effect on [Ca²⁺], mobilization.2. The ed₅₀ value for ACh is 4 (±0.5)· 10⁻⁶M and for ATP 20 (±5)· 10⁻⁶M.3. Addition of ACh and ATP to dissociated chick embryo cells causes rapid accumulation of IP₃.4. The stimulatory effects of ACh and ATP on [Ca²⁺], mobilization and IP₃ rapid formation are both additive.     

Antibodies from Trypanosoma cruzi infected mice recognize the second extracellular loop of the β1-adrenergic and M2-muscarinic receptors and regulate calcium channels in isolated cardiomyocytes

Sera from T. cruzi infected mice were tested in an enzyme immunoassay on peptides corresponding to the second extracellular loops of the –, the ₂-adrenergic receptor and the M2 muscarinic receptor. All sera of mice (4/4) in the acute phase recognized the ₁-adrenergic receptor and the M2 muscarinic receptor peptides but not the ₂-adrenergic receptor peptide. The same peptides were recognized during the chronic phase in half of the mice (6/12). The immunoglobulin fractions of the mice were tested for their activity on L-type Ca^–+ channels of isolated guinea-pig cardiomyocytes using the whole-cell patch clamp technique. The immunoglobulin fractions of acute phase mice were able to activate the Ca^–+ channels by stimulation of the -adrenergic receptors, as assessed by inhibition with propranolol. Those of the chronic phase mice reduced the Ca⁺⁺ current by stimulation of the muscarinic receptors, as assessed by inhibition with atropine.These results confirm the existence of functional epitopes on the second extracellular loops of both receptors. They suggest that, as in humans, the parasite is able to elicit functional autoantibodies against these epitopes. They give evidence that these autoantibodies mediate their physiological effects by modulating the cAMP activated Ca^+– channels.     

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP₃), which operates as an InsP₃-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP₃-sensitive intracellular Ca²⁺ stores, and a sustained phase of Ca²⁺ influx. These events are accompanied by a redistribution of type II InsP₃ receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP₃ receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP₃ receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP₃ receptor aggregation may be a characteristic cellular response to Ca²⁺-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36^M3R cells. Stimulation of these three cell types leads to a reduction in InsP₃ receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP₃ receptors may contribute to the previously observed changes in affinity for InsP₃ in the presence of elevated Ca²⁺ and/or may establish discrete regions within refilled stores with varying capacity to release Ca²⁺ when a subsequent stimulus results in production of InsP₃.     

Lysophosphatidic acid‐induced Ca2+ mobilization in the neural retina of chick embryo

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca²⁺ concentration ([Ca²⁺]_i) with Fura‐2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1–100 μM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca²⁺]_i in a dose‐dependent manner, with an EC₅₀ value of 9.2 μM. The Ca²⁺ rise was also evoked in a Ca²⁺‐free medium, suggesting that release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) was induced by LPA. U‐73122, a blocker of phospholipase C (PLC), inhibited the Ca²⁺ rise to LPA. Pertussis toxin partially inhibited the Ca²⁺ rise to LPA, indicating that G_i/G_o protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca²⁺ rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca²⁺ response to LPA were the same as those of the Ca²⁺ response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca²⁺ rise in the E3 chick retina. Our results show that LPA induces Ca²⁺ mobilization in the embryonic chick retina during neurogenesis. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 495–504, 1999     

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Actions of a tumor-promoting phorbol ester in a cloned rat pituitary cell

Tumor promoters, such as phorbol myristate acetate (PMA), facilitate carcinogenesis by mechanisms that may involve changes in intracellular Ca²⁺ metabolism and distribution of Ca²⁺, as well as activation of a Ca²⁺-and phospholipid-dependent protein kinase, referred to as protein kinase C. We compared the actions of PMA on GH₃ cloned pituitary cells with those of thyrotropin releasing hormone (TRH), an established Ca²⁺-mobilizing agent. The TRH treatment produced a⁴⁵Ca efflux, inhibited⁴⁵Ca uptake, diminished chlortetracycline fluorescence, and stimulated cAMP accumulation and protein synthesis in a Ca²⁺-dependent manner. Like TRH, PMA produced an efflux of⁴⁵Ca and inhibited⁴⁵Ca uptake; however, the phorbol ester stimulated cAMP accumulation and protein synthesis in the absence of external calcium and failed to alter chlortetracycline fluorescence. The TMB-8, a putative inhibitor of the mobilization of membrane-associated Ca²⁺, did not alter PMA-induced stimulation of protein synthesis. The results suggest that PMA-induced changes in Ca²⁺ metabolism are not caused by the mobilization of membrane-associated calcium. Alternative proposals are that PMA (1) inhibits Ca²⁺ influx and/or (2) mobilizes calcium from nonmembranous storage sites. Further study is needed to characterize the mechanism through which tumor-promoting phorbol esters influence Ca²⁺ metabolism and to ascertain the significance of changes in Ca²⁺ metabolism to cellular processes affected by these substances.     

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y₂ receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y₂ receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca²⁺ mobilization indicating that nucleotides cause Ca²⁺ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca²⁺ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca²⁺ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca²⁺ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca²⁺ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca²⁺ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y₂ receptor can cause Ca²⁺ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.     

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.     

Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30–100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3^−/− mice had increased G_q signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca²⁺) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca²⁺ release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3^+/−) had an intermediate increase in maximum Ca²⁺ mobilization. Treatment of PAR3^−/− mice platelets with P2Y₁₂ antagonist (2MeSAMP) did not affect Ca²⁺ mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G_12/13 signaling in response to thrombin was not significantly different between wild type and PAR3^−/− mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca²⁺ mobilization and PKC activation in mouse platelets by physical interaction.