Role of calcium and calmodulin in ciliary stimulation induced by acetylcholine

The goal of this work was to elucidate the molecular events underlying stimulation of ciliary beat frequency (CBF) induced by acetylcholine (ACh) in frog esophagus epithelium. ACh induces a profound increase in CBF and in intracellular Ca(2+) concentration ([Ca(2+)](i)) through M(1) and M(3) muscarinic receptors. The [Ca(2+)](i) slowly decays to the basal level, while CBF stabilizes at an elevated level. These results suggest that ACh triggers Ca(2+)-correlated and -uncorrelated modes of ciliary stimulation. ACh response is abolished by the phospholipase C (PLC) inhibitor U-73122 and by depletion of intracellular Ca(2+) stores but is unaffected by reduction of extracellular Ca(2+) concentration and by blockers of Ca(2+) influx. Therefore, ACh activates PLC and mobilizes Ca(2+) solely from intracellular stores. The calmodulin inhibitors W-7 and calmidazolium attenuate the ACh-induced increase in [Ca(2+)](i) but completely abolish the elevation in CBF. Therefore, elevation of [Ca(2+)](i) is necessary for CBF enhancement but does not lead directly to it. The combined effect of Ca(2+) elevation and of additional factors, presumably mobilized by Ca(2+)-calmodulin, results in a robust CBF enhancement.     

cAMP inhibits IP(3)-dependent Ca(2+) release by preferential activation of cGMP-primed PKG

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca(2+) mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP(3)-dependent Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca(2+) release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP(3) receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP(3)-induced Ca(2+) release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca(2+) release probably reflects inhibition of IP(3) formation.     

Cyclic GMP modulates store-operated calcium entry inducing phosphatidylserine translocation at the surface of megakaryocytic cells

When subjected to stimulation, cells from the vascular compartment show a spontaneous collapse of the plasma membrane phospholipid asymmetry and phosphatidylserine is exposed at the external leaflet. Thus, phosphatidylserine externalization is essential for normal hemostasis and phagocytosis. The mechanism governing the migration of phosphatidylserine to the exoplasmic leaflet is not yet fully understood. We have proposed that store-operated calcium entry (SOCE) constitutes a key step of this process. Here, interaction of [Ca(2+)](i), cAMP and cGMP pathways and phosphatidylserine exposure was examined in human megakaryocytic cells. The membrane permeable cAMP and cGMP analogues, pCPT-cAMP and pCPT-cGMP, enhanced the Ca(2+) signal induced by ionophore and SOCE. Responses to pCPT-cAMP and pCPT-cGMP were independent of protein kinase A, protein kinase G (PKG) or ERK pathways. Inhibition of small G-proteins reduced or abolished the increase of [Ca(2+)](i) induced by pCPT-cAMP or pCPT-cGMP, respectively. pCPT-cGMP but not pCPT-cAMP enhanced the ability of cells to expose phosphatidylserine. This effect was not prevented by the inhibition of PKG or small G-proteins. These results show the differential role of cyclic nucleotides in the Ca(2+)-dependent membrane remodeling. Hence, pCPT-cGMP is another regulatory element for the completion of SOCE-induced phosphatidylserine transmembrane redistribution in HEL cells through a mechanism implicating small G-proteins.     

K(+) channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.     

Acute effects of adenosine triphosphates, cyclic 3',5'-adenosine monophosphates, and follicle-stimulating hormone on cytosolic calcium level in cultured immature rat Ssertoli cells.

The ability of ATP and FSH to induce intracellular calcium [Ca(2+)](i) changes in Sertoli cells is imperfectly understood and reports are conflicting. We have applied the single-cell microfluorometry technique with the calcium probe indo-1 to investigate [Ca(2+)](i) in individual cultured Sertoli cells. When cells were exposed to ATP, cAMP, and FSH, a fast and biphasic increase in [Ca(2+)](i) was obtained in 100%, 70%, and 56% of cells, respectively. Caffeine did not activate Ca(2+) mobilization, while thapsigargin suppressed the peak response. External calcium free-EGTA buffer suppressed the plateau phase, while blockers of voltage-operated Ca(2+) channels did not abolish the response to cAMP and ATP. We conclude that the three messengers mobilized Ca(2+) from intracellular thapsigargin-sensitive stores, which induced a subsequent Ca(2+) influx from the extracellular medium by a voltage-independent Ca(2+) entry. The well-documented mechanisms by which these messengers act on cells support the idea that they release Ca(2+) from smooth endoplasmic reticulum by two different pathways, or that FSH and cAMP first release ATP, which then acts on cells. Among the cells, 77% and 80% responded, respectively, to FSH and cAMP by a delayed long-lasting decrease in [Ca(2+)](i) that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca(2+)](i) from the exchangeable pool to the bound nonexchangeable pools. Involvement of voltage-operated and voltage-independent calcium channels in the response of Sertoli cells to ATP, FSH, and cAMP is discussed.     

Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)](i) transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH.     

Neurotrophin effects on intracellular Ca2+ and force in airway smooth muscle

Neurotrophins [e.g., brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT4)], known to affect neuronal structure and function, are expressed in nonneuronal tissues including the airway. However, their function is unclear. We examined the effect of acute vs. prolonged neurotrophin exposure on regulation of airway smooth muscle (ASM) intracellular Ca(2+) concentration ([Ca(2+)](i)): sarcoplasmic reticulum (SR) Ca(2+) release and Ca(2+) influx (specifically store-operated Ca(2+) entry, SOCE). Human ASM cells were incubated for 30 min in medium (control) or 1 or 10 nM BDNF, NT3, or NT4 (acute exposure) or overnight in 1 nM BDNF, NT3, or NT4 (prolonged exposure) and imaged after loading with the Ca(2+) indicator fura-2 AM. [Ca(2+)](i) responses to ACh, histamine, bradykinin, and caffeine and SOCE following SR Ca(2+) depletion were compared across cell groups. Force measurements were performed in human bronchial strips exposed to neurotrophins. Basal [Ca(2+)](i), peak responses to all agonists, SOCE, and force responses to ACh and histamine were all significantly enhanced by both acute and prolonged BDNF exposure (smaller effect of NT4) but decreased by NT3. Inhibition of the BDNF/NT4 receptor trkB by K252a prevented enhancement of [Ca(2+)](i) responses. ASM cells showed positive immunostaining for BDNF, NT3, NT4, trkB, and trkC (NT3 receptor). These novel data demonstrate that neurotrophins influence ASM [Ca(2+)](i) and force regulation and suggest a potential role for neurotrophins in airway diseases.     

Effects of chronic exercise on calcium signaling in rat vascular endothelium

Chronic exercise enhances endothelium-dependent vasodilating responses. To investigate whether this is due to a change in endothelial Ca(2+) signaling, we examined intracellular Ca(2+) concentration ([Ca(2+)](i)) level in rat aortic endothelium in response to acetylcholine (ACh) or ATP. Four-week-old male Wistar rats were divided into control and exercise groups. The exercised animals ran on a treadmill at a moderate intensity for 60 min/day, 5 day/wk, for 10 wk. Rat aortas were then excised and loaded with fura 2. After the aortas were mounted on a flow chamber, these specimens were observed under an epifluorescence microscope equipped with ratio-imaging capability. Our results showed that 1) chronic exercise increased both ACh- and ATP-induced [Ca(2+)](i) responses; 2) ACh induced heterogeneous [Ca(2+)](i) elevation in individual endothelial cells; and 3) the exercise effect on ACh-evoked endothelial [Ca(2+)](i) elevation was inhibited by the Ca(2+) influx blocker SKF-96365, by a Ca(2+)-free buffer, or by high concentrations of extracellular K(+). We conclude that chronic exercise increases ACh-induced [Ca(2+)](i) elevation in rat aortic endothelium in situ, possibly by facilitating Ca(2+) influx.     

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca(2+) ([Ca(2+)](i)) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca(2+)](i) in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca(2+)](i). Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca(2+)](i). GLP-1 (20 nM) did not affect membrane potential or [Ca(2+)](i). In contrast, acetylcholine (ACh, 100 microM) induced fast membrane depolarization immediately followed by a modest [Ca(2+)](i) increase. When extracellular Ca(2+) was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca(2+)](i) increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP(3)R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca(2+)](i) and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca(2+) through store operated InsP(3)R.     

Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.     

ADP-ribosyl cyclase couples to cyclic AMP signaling in the cardiomyocytes

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR) from beta-NAD(+). In this study, we examined the molecular basis of which beta-adrenergic receptor (betaAR) stimulation induces cADPR formation and characterized cardiac ADPR-cyclase. The results revealed that isoproterenol-mediated increase of [Ca(2+)](i) in rat cardiomyocytes was blocked by pretreatment with a cADPR antagonistic derivative 8-Br-cADPR, a PKA inhibitor H89 or high concentration of ryanodine. Moreover, incubation of ventricular lysates with isoproterenol, forskolin or cAMP resulted in activation of ADPR-cyclase that was inhibited by pretreatment with H89. Supporting the observations, the cADPR antagonist and H89 blocked 8-CPT-cAMP, a cell-permeant cAMP analog-induced increase in [Ca(2+)](i) but not cGMP-mediated increase. Characterization of partially purified cardiac ADPR-cyclase showed a molecular mass of approximately 42 kDa and no cross-activity with CD38 antibodies, and the enzyme activity was inhibited by Zn(2+) but not dithiothreitol. Microinjection of the enzyme into rat cardiomyocytes increased the level of [Ca(2+)](i) in a concentration-dependent manner. The enzyme-mediated increase of [Ca(2+)](i) was blocked by the cADPR antagonist. These findings suggest that betaAR-mediated regulation of [Ca(2+)](i) in rat cardiomyocytes is primed by activation of cardiac ADPR-cyclase via cAMP/PKA signaling and that cardiac ADPR-cyclase differs from CD38 in biochemical and immunological properties.     

Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes

Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 microM cAMP or 10 microM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.     

cGMP stimulates endoplasmic reticulum Ca(2+)-ATPase in vascular endothelial cells

Calcium is a crucial regulator of many physiological processes such as cell growth, division, differentiation, cell death and apoptosis. In this study, we examined the effect of cGMP on agonist-induced [Ca(2+)](i) transient in isolated rat aortic endothelial cells. 100 microM ATP was applied to the cells bathed in a Ca(2+)-free physiological solution to induce a [Ca(2+)](i) transient that was caused by Ca(2+) release from intracellular stores. cGMP, which was applied after [Ca(2+)](i) reached its peak level, accelerated the falling phase of [Ca(2+)](i) transient. Pre-treatment of the cells with CPA abolished the accelerating effect of cGMP on the falling phase of [Ca(2+)](i) transient. The effect of cGMP was reversed by KT5823, a highly specific inhibitor of protein kinase G. Taken together, these data suggest that cGMP may reduce [Ca(2+)](i) level by promoting Ca(2+) uptake through sarcoplasmic/endoplasmic reticulum ATPase and that the effect of cGMP may be mediated by protein kinase G.     

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs.

Transient increases, or oscillations, of cytoplasmic free Ca(2+) concentration, [Ca(2+)](i), occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca(2+) is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca(2+) signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP(3)) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca(2+)](i) rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP(3) levels, all almost doubling before the explosive increase of [Ca(2+)](i); (2) most of the rise in IP(3) occurred after the Ca(2+) peak; IP(3) production could also be induced by the artificial elevation of [Ca(2+)](i), suggesting the large increase in IP(3) is a consequence, rather than a cause, of the Ca(2+) transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP(3) and the [Ca(2+)](i) increase without the delay of Ca(2+) transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca(2+) transients by stimulating IP(3) production during fertilization of sea urchin eggs.     

Cytosolic [Ca2+] transients in dictyostelium discoideum depend on the filling state of internal stores and on an active sarco/endoplasmic reticulum calcium ATPase (SERCA) Ca2+ pump

Stimulation of Dictyostelium discoideum with cAMP evokes a change of the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). We analyzed the role of the filling state of Ca(2+) stores for the [Ca(2+)] transient. Parameters tested were the height of the [Ca(2+)](i) elevation and the percentage of responding amoebae. After loading stores with Ca(2+), cAMP induced a [Ca(2+)](i) transient in many cells. Without prior loading, cAMP evoked a [Ca(2+)](i) change in a few cells only. This indicates that the [Ca(2+)](i) elevation is not mediated exclusively by Ca(2+) influx but also by Ca(2+) release from stores. Reducing the Ca(2+) content of the stores by EGTA preincubation led to a cAMP-activated [Ca(2+)](i) increase even at low extracellular [Ca(2+)]. Moreover, the addition of Ca(2+) itself elicited a capacitative [Ca(2+)](i) elevation. This effect was not observed when stores were emptied by the standard technique of inhibiting internal Ca(2+) pumps with 2,5-di-(t-butyl)-1,4-hydroquinone. Therefore, in Dictyostelium, an active internal Ca(2+)-ATPase is absolutely required to allow for Ca(2+) entry. No influence of the filling state of stores on Ca(2+) influx characteristics was found by the Mn(2+)-quenching technique, which monitors the rate of Ca(2+) entry. Both basal and cAMP-activated Mn(2+) influx rates were similar in control cells and cells with empty stores. By contrast, determination of extracellular free Ca(2+) concentration ([Ca(2+)](e)) changes, which represent the sum of Ca(2+) influx and efflux, revealed a higher rate of [Ca(2+)](e) decrease in EGTA-treated than in control amoebae. We conclude that emptying of Ca(2+) stores does not change the rate of Ca(2+) entry but results in inhibition of the plasma membrane Ca(2+)-ATPase. Furthermore, the activities of the Ca(2+) transport ATPases of the stores are of crucial importance for the regulation of [Ca(2+)](i) changes.     

Effect of diindolylmethane on Ca(2+) homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 μM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 μM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 μM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.     

Low [Mg(2+)](o) induces contraction and [Ca(2+)](i) rises in cerebral arteries: roles of ca(2+), PKC, and PI3

Removal of extracellular Ca(2+) concentration ([Ca(2+)](o)) and pretreatment of canine basilar arterial rings with either an antagonist of voltage-gated Ca(2+) channels (verapamil), a selective antagonist of the sarcoplasmic reticulum Ca(2+) pump [thapsigargin (TSG)], caffeine plus a specific antagonist of ryanodine-sensitive Ca(2+) release (ryanodine), or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]- mediated Ca(2+) release antagonist (heparin) markedly attenuates low extracellular Mg(2+) concentration ([Mg(2+)](o))-induced contractions. Low [Mg(2+)](o)-induced contractions are significantly inhibited by pretreatment of the vessels with G?-6976 [a protein kinase C-alpha (PKC-alpha)- and PKC-betaI-selective antagonist], bisindolylmaleimide I (Bis, a specific antagonist of PKC), and wortmannin or LY-294002 [selective antagonists of phosphatidylinositol-3 kinases (PI3Ks)]. These antagonists were also found to relax arterial contractions induced by low [Mg(2+)](o) in a concentration-dependent manner. The absence of [Ca(2+)](o) and preincubation of the cells with verapamil, TSG, heparin, or caffeine plus ryanodine markedly attenuates the transient and sustained elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by low-[Mg(2+)](o) medium. Low [Mg(2+)](o)-produced increases in [Ca(2+)](i) are also suppressed markedly in the presence of G?-6976, Bis, wortmannin, or LY-294002. The present study suggests that both Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from intracellular stores [both Ins(1,4,5)P(3) sensitive and ryanodine sensitive] play important roles in low-[Mg(2+)](o) medium-induced contractions of isolated canine basilar arteries. Such contractions are clearly associated with activation of PKC isoforms and PI3Ks.     

Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. A mechanism for the synergistic effects of cAMP on Ca2+ signaling.

Acetylcholine-evoked secretion from the parotid gland is substantially potentiated by cAMP-raising agonists. A potential locus for the action of cAMP is the intracellular signaling pathway resulting in elevated cytosolic calcium levels ([Ca(2+)](i)). This hypothesis was tested in mouse parotid acinar cells. Forskolin dramatically potentiated the carbachol-evoked increase in [Ca(2+)](i), converted oscillatory [Ca(2+)](i) changes into a sustained [Ca(2+)](i) increase, and caused subthreshold concentrations of carbachol to increase [Ca(2+)](i) measurably. This potentiation was found to be independent of Ca(2+) entry and inositol 1,4,5-trisphosphate (InsP(3)) production, suggesting that cAMP-mediated effects on Ca(2+) release was the major underlying mechanism. Consistent with this hypothesis, dibutyryl cAMP dramatically potentiated InsP(3)-evoked Ca(2+) release from streptolysin-O-permeabilized cells. Furthermore, type II InsP(3) receptors (InsP(3)R) were shown to be directly phosphorylated by a protein kinase A (PKA)-mediated mechanism after treatment with forskolin. In contrast, no evidence was obtained to support direct PKA-mediated activation of ryanodine receptors (RyRs). However, inhibition of RyRs in intact cells, demonstrated a role for RyRs in propagating Ca(2+) oscillations and amplifying potentiated Ca(2+) release from InsP(3)Rs. These data indicate that potentiation of Ca(2+) release is primarily the result of PKA-mediated phosphorylation of InsP(3)Rs, and may largely explain the synergistic relationship between cAMP-raising agonists and acetylcholine-evoked secretion in the parotid. In addition, this report supports the emerging consensus that phosphorylation at the level of the Ca(2+) release machinery is a broadly important mechanism by which cells can regulate Ca(2+)-mediated processes.     

ACh对人垂体腺瘤细胞内蛋白激酶C、胞内游离钙及cAMP/cGMP的影响

我们曾发现ACh可明显地抑制垂体腺瘤细胞的增殖代谢,为深入探讨ACh抑制垂体腺瘤细胞增殖作用的机制,观察了ACh作用后垂体腺瘤细胞内蛋白激酶C(PKC)、[Ca^2 ]i及cAMP／cGMP的变化。结果发现：(1)与空白处理组相比,使用PKC的激动剂PMA处理培养的人垂体腺瘤细胞时可使胞浆、胞膜和细胞总PKC活性浓度均升高,但ACh(10μmol/L)作用15min后,胞浆、胞膜和细胞总PKC活性均下降,且此作用可被阿托品阻断;(2)ACh(10μmol/L)作用于单个人垂体腺瘤细胞后,立即使垂体腺瘤细胞[Ca^2 ]i相对水平降低,但此作用可被阿托品阻断;(3)ACh作用于人垂体腺瘤细胞15min后,胞内cAMP水平均明显升高,而cGMP没有改变。该结果为探讨ACh抑制垂体腺瘤细胞增殖的分子机制提供了重要线索,同时提示,ACh对垂体瘤细胞增殖分化的调控作用是细胞内多信息系统相互整合的结果。