Conjunctival goblet cell secretion stimulated by leukotrienes is reduced by resolvins D1 and E1 to promote resolution of inflammation

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.     

Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity.     

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.     

ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling

We tested whether the respective angiotensin type 1 (AT(1)) and 2 (AT(2)) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca(2+)](i)) suppression induced by ANG II. ANG II partially reversed the increase in [Ca(2+)](i) generated by cyclopiazonic acid (CPA; 10(-5) M), acetylcholine (ACh; 10(-5) M), or bradykinin (BK; 10(-7) M). Losartan (10(-5) M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT(2) receptor blockade with PD-123319 (10(-8) M) augmented the suppression of endothelial [Ca(2+)](i) responses. Similarly, preactivation with the AT(2) receptor agonist CGP-42112A (10(-8) M) prevented AT(1) receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca(2+)](i). In contrast to endothelial [Ca(2+)](i) suppression by ANG II, pericyte [Ca(2+)](i) exhibited typical peak and plateau [Ca(2+)](i) responses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT(2) receptors were blocked with PD-123319. Similarly, AT(2) receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10(-5) M) showed no AT(1)-like action to constrict microperfused DVR or increase pericyte [Ca(2+)](i). We conclude that ANG II suppression of endothelial [Ca(2+)](i) and stimulation of pericyte [Ca(2+)](i) is mediated by AT(1) or AT(1)-like receptors. Furthermore, AT(2) receptor activation opposes ANG II-induced endothelial [Ca(2+)](i) suppression and abrogates ANG II-induced DVR vasoconstriction.     

Intracellular angiotensin II activates rat myometrium

Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.     

Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells

ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP-induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.     

Mode of action of ANG II on ion transport in guinea pig distal colon

The effect of ANG II on mucosal ion transport and localization of ANG type 1 receptor (AT(1)R) in the guinea pig distal colon was investigated. Submucosal/mucosal segments were mounted in Ussing flux chambers, and short-circuit current (I(sc)) was measured as an index of ion transport. Serosal addition of ANG II produced a concentration-dependent (10(-9)-10(-5) M) increase in I(sc). The maximal response was observed at 10(-6) M; the increase in I(sc) was 164.4 +/- 11.8 microA/cm(2). The ANG II (10(-6) M)-evoked response was mainly due to Cl(-) secretion. Tetrodotoxin, atropine, the neurokinin type 1 receptor antagonist FK-888, and piroxicam significantly reduced the ANG II (10(-6) M)-evoked response to 28, 45, 58, and 16% of control, respectively. Pretreatment with prostaglandin E(2) (10(-5) M) resulted in a threefold increase in the ANG II-evoked response. The AT(1)R antagonist FR-130739 completely blocked ANG II (10(-6) M)-evoked responses, whereas the ANG type 2 receptor antagonist PD-123319 had no effect. Localization of AT(1)R was determined by immunohistochemistry. In the immunohistochemical study, AT(1)R-immunopositive cells were distributed clearly in enteric nerves and moderately in surface epithelial cells. These results suggest that ANG II-evoked electrogenic Cl(-) secretion may involve submucosal cholinergic and tachykinergic neurons and prostanoid synthesis pathways through AT(1)R on the submucosal plexus and surface epithelial cells in guinea pig distal colon.     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.     

Inhibition of calcium signaling in descending vasa recta endothelia by ANG II

The intracellular calcium ([Ca(2+)](i)) response of outer medullary descending vasa recta (OMDVR) endothelia to ANG II was examined in fura 2-loaded vessels. Abluminal ANG II (10(-8) M) caused [Ca(2+)](i) to fall in proportion to the resting [Ca(2+)](i) (r = 0. 82) of the endothelium. ANG II (10(-8) M) also inhibited both phases of the [Ca(2+)](i) response generated by bradykinin (BK, 10(-7) M), 835 +/- 201 versus 159 +/- 30 nM (peak phase) and 169 +/- 26 versus 103 +/- 14 nM (plateau phase) (means +/- SE). Luminal ANG II reduced BK (10(-7) M)-stimulated plateau [Ca(2+)](i) from 180 +/- 40 to 134 +/- 22 nM without causing vasoconstriction. Abluminal ANG II added to the bath after luminal application further reduced [Ca(2+)](i) to 113 +/- 9 nM and constricted the vessels. After thapsigargin (TG) pretreatment, ANG II (10(-8) M) caused [Ca(2+)](i) to fall from 352 +/- 149 to 105 +/- 37 nM. This effect occurred at a threshold ANG II concentration of 10(-10) M and was maximal at 10(-8) M. ANG II inhibited both the rate of Ca(2+) entry into [Ca(2+)](i)-depleted endothelia and the rate of Mn(2+) entry into [Ca(2+)](i)-replete endothelia. In contrast, ANG II raised [Ca(2+)](i) in the medullary thick ascending limb and outer medullary collecting duct, increasing [Ca(2+)](i) from baselines of 99 +/- 33 and 53 +/- 11 to peaks of 200 +/- 47 and 65 +/- 11 nM, respectively. We conclude that OMDVR endothelia are unlikely to be the source of ANG II-stimulated NO production in the medulla but that interbundle nephrons might release Ca(2+)-dependent vasodilators to modulate vasomotor tone in vascular bundles.     

Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2(+)-induced Ca2+ release.

Receptor-mediated inositol 1,4,5-trisphosphate (Ins-(1,4,5)P3) generation evokes fluctuations in the cytoplasmic Ca2+ concentration ([Ca2+]i). Intracellular Ca2+ infusion into single mouse pancreatic acinar cells mimicks the effect of external acetylcholine (ACh) or internal Ins(1,4,5)P3 application by evoking repetitive Ca2+ release monitored by Ca2(+)-activated Cl- current. Intracellular infusion of the Ins(1,4,5)P3 receptor antagonist heparin fails to inhibit Ca2+ spiking caused by Ca2+ infusion, but blocks ACh- and Ins(1,4,5)P3-evoked Ca2+ oscillations. Caffeine (1 mM), a potentiator of Ca2(+)-induced Ca2+ release, evokes Ca2+ spiking during subthreshold intracellular Ca2+ infusion. These results indicate that ACh-evoked Ca2+ oscillations are due to pulses of Ca2+ release through a caffeine-sensitive channel triggered by a small steady Ins(1,4,5)P3-evoked Ca2+ flow.     

Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.     

Functionally separate intracellular Ca2+ stores in smooth muscle

In smooth muscle, release via the inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) controls oscillatory and steady-state cytosolic Ca(2+) concentrations ([Ca(2+)](c)). The interplay between the two receptors, itself determined by their organization on the SR, establishes the time course and spatial arrangement of the Ca(2+) signal. Whether or not the receptors are co-localized or distanced from each other on the same store or whether they exist on separate stores will significantly affect the Ca(2+) signal produced by the SR. To date these matters remain unresolved. The functional arrangement of the RyR and Ins(1,4,5)P(3)R on the SR has now been examined in isolated single voltage-clamped colonic myocytes. Depletion of the ryanodine-sensitive store, by repeated application of caffeine, in the presence of ryanodine, abolished the response to Ins(1,4,5)P(3), suggesting that Ins(1,4,5)P(3)R and RyR share a common Ca(2+) store. Ca(2+) release from the Ins(1,4,5)P(3)R did not activate Ca(2+)-induced Ca(2+) release at the RyR. Depletion of the Ins(1,4,5)P(3)-sensitive store, by the removal of external Ca(2+), on the other hand, caused only a small decrease ( approximately 26%) in caffeine-evoked Ca(2+) transients, suggesting that not all RyR exist on the common store shared with Ins(1,4,5)P(3)R. Dependence of the stores on external Ca(2+) for replenishment also differed; removal of external Ca(2+) depleted the Ins(1,4,5)P(3)-sensitive store but caused only a slight reduction in caffeine-evoked transients mediated at RyR. Different mechanisms are presumably responsible for the refilling of each store. Refilling of both Ins(1,4,5)P(3)-sensitive and caffeine-sensitive Ca(2+) stores was inhibited by each of the SR Ca(2+) ATPase inhibitors thapsigargin and cyclopiazonic acid. These results may be explained by the existence of two functionally distinct Ca(2+) stores; the first expressing only RyR and refilled from [Ca(2+)](c), the second expressing both Ins(1,4,5)P(3)R and RyR and dependent upon external Ca(2+) for refilling.     

Dual role of PKC in modulating pharmacomechanical coupling in fetal and adult cerebral arteries

This study tested the hypothesis that protein kinase C (PKC) has dual regulation on norepinephrine (NE)-mediated inositol 1,4, 5-trisphosphate [Ins (1,4,5)P(3)] pathway and vasoconstriction in cerebral arteries from near-term fetal ( approximately 140 gestational days) and adult sheep. Basal PKC activity values (%membrane bound) in fetal and adult cerebral arteries were 38 +/- 4% and 32 +/- 4%, respectively. In vessels of both age groups, the PKC isoforms alpha, beta(I), beta(II), and delta were relatively abundant. In contrast, compared with the adult, cerebral arteries of the fetus had low levels of PKC-epsilon. In response to 10(-4) M phorbol 12,13-dibutyrate (PDBu; PKC agonist), PKC activity in both fetal and adult cerebral arteries increased 40-50%. After NE stimulation, PKC activation with PDBu exerted negative feedback on Ins(1,4,5)P(3) and intracellular Ca(2+) concentration ([Ca(2+)](i)) in arteries of both age groups. In turn, PKC inhibition with staurosporine resulted in augmented NE-induced Ins(1,4,5)P(3) and [Ca(2+)](i) responses in adult, but not fetal, cerebral arteries. In adult tissues, PKC stimulation by PDBu increased vascular tone, but not [Ca(2+)](i). In contrast, in the fetal artery, PKC stimulation was associated with an increase in both tone and [Ca(2+)](i). In the presence of zero extracellular [Ca(2+)], these PDBu-induced responses were absent in the fetal vessel, whereas they remained unchanged in the adult. We conclude that, although basal PKC activity was similar in fetal and adult cerebral arteries, PKC's role in NE-mediated pharmacomechanical coupling differed significantly in the two age groups. In both fetal and adult cerebral arteries, PKC modulation of NE-induced signal transduction responses would appear to play a significant role in the regulation of vascular tone. The mechanisms differ in the two age groups, however, and this probably relates, in part, to the relative lack of PKC-epsilon in fetal vessels.     

Angiotensin IV-mediated pulmonary artery vasorelaxation is due to endothelial intracellular calcium release

Angiotensin (ANG) IV stimulation of pulmonary artery (PA) endothelial cells (PAECs) but not of PA smooth muscle cells (PASMCs) resulted in significant increased production of cGMP in PASMCs. ANG IV receptors are not present in PASMCs, and PASMC nitric oxide synthase activity was not altered by ANG IV. ANG IV caused a dose-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PAs, and this response was blocked by the ANG IV receptor antagonist divalinal ANG IV but not by ANG II type 1 and 2 receptor blockers. ANG IV receptor-mediated increased intracellular Ca(2+) concentration ([Ca(2+)](i)) release from intracellular stores in PAECs was blocked by divalinal ANG IV as well as by the G protein, phospholipase C, and phosphoinositide (PI) 3-kinase inhibitors guanosine 5'-O-(2-thiodiphosphate), U-73122, and LY-294002, respectively, and was regulated by both PI 3-kinase- and ryanodine-sensitive Ca(2+) stores. Basal and ANG IV-mediated vasorelaxation of endothelium-denuded PAs was restored by exogenous PAECs but not by exogenous PAECs pretreated with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. These results demonstrate that ANG IV-mediated vasodilation of PAs is endothelium dependent and regulated by [Ca(2+)](i) release through receptor-coupled G protein-phospholipase C-PI 3-kinase signaling mechanisms.     

Enhanced [Ca2+]i in renal arterial smooth muscle cells of pregnant rats with reduced uterine perfusion pressure

Reduction of uterine perfusion pressure (RUPP) during late pregnancy has been suggested to trigger increases in renal vascular resistance and lead to hypertension of pregnancy. We investigated whether the increased renal vascular resistance associated with RUPP in late pregnancy reflects increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and contraction of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of normal pregnant Sprague-Dawley rats and a rat model of RUPP during late pregnancy. The cells were loaded with fura 2 and both cell length and [Ca(2+)](i) were measured. In cells of normal pregnant rats incubated in Hanks' solution (1 mM Ca(2+)), ANG II (10(-7) M) caused an initial increase in [Ca(2+)](i) to 414 +/- 13 nM, a maintained increase to 149 +/- 8 nM, and 21 +/- 1% cell contraction. In RUPP rats, the initial ANG II-induced [Ca(2+)](i) (431 +/- 18 nM) was not different from pregnant rats, but both the maintained [Ca(2+)](i) (225 +/- 9 nM) and cell contraction (48 +/- 2%) were increased. Membrane depolarization by 51 mM KCl and the Ca(2+) channel agonist BAY K 8644 (10(-6) M), which stimulate Ca(2+) entry from the extracellular space, caused maintained increases in [Ca(2+)](i) and cell contraction that were greater in RUPP rats than control pregnant rats. In Ca(2+)-free (2 mM EGTA) Hanks' solution, the ANG II- and caffeine (10 mM)-induced [Ca(2+)](i) transient and cell contraction were not different between normal pregnant and RUPP rats, suggesting no difference in Ca(2+) release from the intracellular stores. The enhanced maintained ANG II-, KCl- and BAY K 8644-induced [Ca(2+)](i) and cell contraction in RUPP rats compared with normal pregnant rats suggest enhanced Ca(2+) entry mechanisms of smooth muscle contraction in resistance renal arteries and may explain the increased renal vascular resistance associated with hypertension of pregnancy.     

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.     

Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin

Bovine adrenocortical cells express bTREK-1 K(+) (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca(2+) and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca(2+)-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca(2+) ionophore ionomycin (1 μM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5-10 μM) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP](i). BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca(2+) concentration ([Ca(2+)](i)) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 ± 4.5% (n=11) and 59.3 ± 8.4% (n=4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP(2)) analog DiC(8)PI(4,5)P(2) in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability (P(o)) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca(2+) (10-35 μM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K(+) channels by a Ca(2+)-dependent mechanism that does not require the depletion of membrane-associated PIP(2). They further indicate that the Ca(2+) source is located in close proximity within a "Ca(2+) nanodomain" of bTREK-1 channels, where [Ca(2+)](i) may reach concentrations of >10 μM. bTREK-1 is the first two-pore K(+) channel shown to be inhibited by Ca(2+) through activation of a G protein-coupled receptor.     

肾上腺髓质素降低培养海马神经元胞内游离钙离子浓度

经荧光探针Fluo 3-AM标记细胞内游离钙后,用激光共聚焦显微镜检测肾上腺髓质素(adrenomedullin,ADM)对原代培养大鼠海马神经元内游离钙浓度([Ca^2 ]1)的影响。实验结果如下：(1)ADM(0．01-1．0μmol／L)浓度依赖性地降低细胞内钙浓度。(2)降钙素基因相关肽受体阻断剂(calcitonin gene-related peptide,CGRP8-37)预处理可部分抑制ADM的效应。(3)ADM可显著抑制高钾引起的[Ca^2 ]1增加。(4)ADM可显著抑制三磷酸肌醇(inositol 1,4,5-trisphosphate,IP3)引起的内钙释放,而对兰尼定(ryanodine)引起的内钙释放无显著影响。以上结果提示,ADM降低培养海马神经元内游离钙浓度,此作用与其抑制IP,引起的内钙释放有关,ADM对静息状态下的Ca^2 内流无影响,但可显著抑制高钾引起的Ca^2 内流,CGRP受体介导了ADM的上述效应。     

Membrane potential controls calcium entry into descending vasa recta pericytes

We tested the hypothesis that constriction of descending vasa recta (DVR) is mediated by voltage-gated calcium entry. K(+) channel blockade with BaCl(2) (1 mM) or TEACl (30 mM) depolarized DVR smooth muscle/pericytes and constricted in vitro-perfused vessels. Pericyte depolarization by 100 mM extracellular KCl constricted DVR and increased pericyte intracellular Ca(2+) ([Ca(2+)](i)). The K(ATP) channel opener pinacidil (10(-7)-10(-4) M) hyperpolarized resting pericytes, repolarized pericytes previously depolarized by ANG II (10(-8) M), and vasodilated DVR. The DVR vasodilator bradykinin (10(-7) M) also reversed ANG II depolarization. The L-type Ca(2+) channel blocker diltiazem vasodilated ANG II (10(-8) M)- or KCl (100 mM)-preconstricted DVR, and the L-type agonist BayK 8644 constricted DVR. The plateau phase of the pericyte [Ca(2+)](i) response to ANG II was inhibited by diltiazem. These data support the conclusion that DVR vasoreactivity is controlled through variation of membrane potential and voltage-gated Ca(2+) entry into the pericyte cytoplasm.     

Ca2+ requirements for cerebellar long-term synaptic depression: role for a postsynaptic leaky integrator

Photolysis of a caged Ca(2+) compound was used to characterize the dependence of cerebellar long-term synaptic depression (LTD) on postsynaptic Ca(2+) concentration ([Ca(2+)](i)). Elevating [Ca(2+)](i) was sufficient to induce LTD without requiring any of the other signals produced by synaptic activity. A sigmoidal relationship between [Ca(2+)](i) and LTD indicated a highly cooperative triggering of LTD by Ca(2+). The duration of the rise in [Ca(2+)](i) influenced the apparent Ca(2+) affinity of LTD, and this time-dependent behavior could be described by a leaky integrator process with a time constant of 0.6 s. A computational model, based on a positive-feedback cycle that includes protein kinase C and MAP kinase, was capable of simulating these properties of Ca(2+)-triggered LTD. Disrupting this cycle experimentally also produced the predicted changes in the Ca(2+) dependence of LTD. We conclude that LTD arises from a mechanism that integrates postsynaptic Ca(2+) signals and that this integration may be produced by the positive-feedback cycle.