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1.
In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca2+ concentration ([Ca2+]i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca2+]i. Low concentrations of ATP (<10 µM) induced intracellular Ca2+ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca2+]i rise and increased the exocytosis rate sevenfold. The contribution of Ca2+ or cAMP pathways to exocytosis was tested by using the Ca2+ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca2+]i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca2+ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate  相似文献   

2.
Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR)Ca2+-ATPase, and this inhibition is relieved bycAMP-dependent protein kinase (PKA)-mediated phosphorylation. The roleof PLB in regulating Ca2+ release throughryanodine-sensitive Ca2+ release channels, measured asCa2+ sparks, was examined using smooth muscle cells ofcerebral arteries from PLB-deficient ("knockout") mice(PLB-KO). Ca2+ sparks were monitored opticallyusing the fluorescent Ca2+ indicator fluo 3 or electricallyby measuring transient large-conductance Ca2+-activatedK+ (BK) channel currents activated by Ca2+sparks. Basal Ca2+ spark and transient BK current frequencywere elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, anactivator of adenylyl cyclase, increased the frequency ofCa2+ sparks and transient BK currents in cerebral arteriesfrom control mice. However, forskolin had little effect on thefrequency of Ca2+ sparks and transient BK currents fromPLB-KO cerebral arteries. Forskolin or PLB-KO increased SRCa2+ load, as measured by caffeine-induced Ca2+transients. This study provides the first evidence that PLB is criticalfor frequency modulation of Ca2+ sparks and associated BKcurrents by PKA in smooth muscle.

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3.
Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular Ca2+ concentration ([Ca2+]i) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [Ca2+]i was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3–10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [Ca2+]i in secretin- or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [Ca2+]i in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3–10 mM concentrations significantly reduced fluid secretion and decreased [Ca2+]i in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [Ca2+]i response to ACh as well as the [Ca2+]i elevation induced by thapsigargin, suggesting that they inhibit Ca2+ influx. Removal of extracellular Ca2+ reduced [Ca2+]i in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [Ca2+]i in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane Ca2+ channel. intracellular calcium; acetylcholine  相似文献   

4.
Caffeine has been shown to increase the Ca2+ release frequency (Ca2+ sparks) from the sarcoplasmic reticulum (SR) through ryanodine-sensitive stores and relax gastric fundus smooth muscle. Increased Ca2+ store refilling increases the frequency of Ca2+ release events and store refilling is enhanced by CaM kinase II (CaMKII) phosphorylation of phospholamban (PLB). These findings suggest that transient, localized Ca2+ release events from the SR may activate CaMKII and contribute to relaxation by enhancing store refilling due to PLB Thr17 phosphorylation. To investigate this possibility, we examined the effects of caffeine on CaMKII, muscle tone, and PLB phosphorylation in murine gastric fundus smooth muscle. Caffeine (1 mM) hyperpolarized and relaxed murine gastric fundus smooth muscle and activated CaMKII. Ryanodine, tetracaine, or cyclopiazonic acid each prevented CaMKII activation and significantly inhibited caffeine-induced relaxation. The large-conductance Ca2+-activated K+ channel blocker iberiotoxin, but not apamin, partially inhibited caffeine-induced relaxation. Caffeine-induced CaMKII activation increased PLB Thr17, but not PLB Ser16 phosphorylation. 3-Isobutyl-1-methylxanthine increased PLB Ser16 phosphorylation, but not PLB Thr17 phosphorylation. The CaMKII inhibitor KN-93 inhibited caffeine-induced relaxation and PLB Thr17 phosphorylation. These results show that caffeine-induced CaMKII activation and PLB phosphorylation play a role in the relaxation of gastric fundus smooth muscles. Ca2+/CaM-dependent protein kinase II  相似文献   

5.
Although dilated cardiomyopathy (DCM) is known to result in cardiac contractile dysfunction, the underlying mechanisms are unclear. The sarcoplasmic reticulum (SR) is the main regulator of intracellular Ca2+ required for cardiac contraction and relaxation. We therefore hypothesized that abnormalities in both SR function and regulation will contribute to cardiac contractile dysfunction of the J2N-k cardiomyopathic hamster, an appropriate model of DCM. Echocardiographic assessment indicated contractile dysfunction, because the ejection fraction, fractional shortening, cardiac output, and heart rate were all significantly reduced in J2N-k hamsters compared with controls. Depressed cardiac function was associated with decreased cardiac SR Ca2+ uptake in the cardiomyopathic hamsters. Reduced SR Ca2+ uptake could be further linked to a decrease in the expression of the SR Ca2+-ATPase and cAMP-dependent protein kinase (PKA)-mediated phospholamban (PLB) phosphorylation at serine-16. Depressed PLB phosphorylation was paralleled with a reduction in the activity of SR-associated PKA, as well as an elevation in protein phosphatase activity in J2N-k hamster. The results of this study suggest that an alteration in SR function and its regulation contribute to cardiac contractile dysfunction in the J2N-k cardiomyopathic hamster. sarcoplasmic reticulum; cardiomyopathy; cAMP-dependent protein kinase; Ca2+/calmodulin-dependent protein kinase; sarco(endo)plasmic reticulum ATPase; phospholamban  相似文献   

6.
The intent of this work was to evaluate the role of cAMP inregulation of ciliary activity in frog mucociliary epithelium and toexamine the possibility of cross talk between the cAMP- andCa2+-dependent pathways in thatregulation. Forskolin and dibutyryl cAMP induced strong transientintracellular Ca2+ concentration([Ca2+]i)elevation and strong ciliary beat frequency enhancement with prolongedstabilization at an elevated plateau. The response was not affected byreduction of extracellular Ca2+concentration. The elevation in[Ca2+]iwas canceled by pretreatment with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Underthose experimental conditions, forskolin raised the beat frequency to amoderately elevated plateau, whereas the initial strong rise infrequency was completely abolished. All effects were canceled by H-89,a selective protein kinase A (PKA) inhibitor. The results suggest adual role for PKA in ciliary regulation. PKA releasesCa2+ from intracellular stores,strongly activating ciliary beating, and, concurrently, producesmoderate prolonged enhancement of the beat frequency by aCa2+-independent mechanism.

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7.
The effects of the cAMP pathway on theCa2+ response elicited byphospholipase C-coupled receptor stimulations were studied in ratparotid cells. Although 1 µM isoproterenol (Iso) itself had no effect on the cytosolicCa2+ concentration, thepretreatment with Iso potentiatedCa2+ responses evoked byphenylephrine. The potentiating effect of Iso was attributed to ashifting of the concentration-response curves of phenylephrine to theleft and an increase in the maximal response. Half-maximal potentiationoccurred at 3 nM Iso. Iso also potentiated theCa2+ response elicited bycarbachol. The potentiating effect of Iso was mimicked by forskolin (10 µM) and dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) and was blocked by 10 µM H-89. Iso potentiated thephenylephrine-induced Ca2+response in the absence of extracellularCa2+, but Iso did not increase theinositol trisphosphate (IP3)production induced by phenylephrine. These results suggest that thepotentiation of the Ca2+ responsecan be attributed to a sensitization ofIP3 receptors by cAMP-dependentprotein kinase.

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8.
Direct effects of parathyroid hormone (PTH) on calcium uptake byisolated rat duodenal cell preparations enriched in enterocytes wereinvestigated. PTH significantly stimulated enterocyte45Ca2+influx in a time-dependent (1-10 min) manner and at all doses tested (2 × 1013 to107 M). TheCa2+ channel antagonists verapamil(10 µM) and nitrendipine (1 µM) completely blocked the stimulationof Ca2+ influx by the hormone(108 M). PTH markedlyincreased cAMP levels in rat duodenal cells (88, 167, and 67%, after1, 2, and 3 min, respectively). In agreement with these observations,forskolin (adenylate cyclase activator), dibutyryl adenosine3',5'-cyclic monophosphate (DBcAMP), and Sp-cAMPS (cAMPanalogs) mimicked, whereas Rp-cAMPS (cAMP antagonist) suppressed PTHand DBcAMP activation of enterocyte calcium uptake. Furthermore, theeffects of DBcAMP were abolished by nitrendipine. These results showdirect rapid effects of PTH on duodenal cells'Ca2+ influx, which involve theactivation of a dihydropyridine-sensitive Ca2+ influx pathway and the cAMPsecond messenger system.

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9.
Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (±dP/dtmax) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca2+ homeostasis that determines the contractile function. Particular emphasis was given to Ca2+ handling by ryanodine receptor (RyR) and Na+-Ca2+ exchange (NCX). 45Ca2+ fluxes via the RyR, NCX, and Ca2+-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca2+ by these Ca2+ handling proteins with the overall Ca2+ homeostasis by determining the Ca2+ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca2+ concentration ([Ca2+]i) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca2+ handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca2+ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca2+ through RyR and removal of Ca2+ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx. ovariectomy  相似文献   

10.
During hypoxia, the level of adenosine in the carotid bodies increases as a result of ATP catabolism and adenosine efflux via adenosine transporters. Using Ca2+ imaging, we found that adenosine, acting via A2A receptors, triggered a rise in cytoplasmic [Ca2+] ([Ca2+]i) in type I (glomus) cells of rat carotid bodies. The adenosine response could be mimicked by forskolin (but not its inactive analog), and could be abolished by the PKA inhibitor H89. Simultaneous measurements of membrane potential (perforated patch recording) and [Ca2+]i showed that the adenosine-mediated [Ca2+]i rise was accompanied by depolarization. Ni2+, a voltage-gated Ca2+ channel (VGCC) blocker, abolished the adenosine-mediated [Ca2+]i rise. Although adenosine was reported to inhibit a 4-aminopyridine (4-AP)-sensitive K+ current, 4-AP failed to trigger any [Ca2+]i rise, or to attenuate the adenosine response. In contrast, anandamide, an inhibitor of the TWIK-related acid-sensitive K+-1 (TASK-1) channels, triggered depolarization and [Ca2+]i rise. The adenosine response was attenuated by anandamide but not by tetraethylammonium. Our results suggest that adenosine, acting via the adenylate cyclase and PKA pathways, inhibits the TASK-1 K+ channels. This leads to depolarization and activation of Ca2+ entry via VGCC. This excitatory action of adenosine on type I cells may contribute to the chemosensitivity of the carotid body during hypoxia. O2 sensing; A2A receptor; cAMP; protein kinase A; TWIK-related acid-sensitive K+ channel  相似文献   

11.
In the presentstudy, we examined the ability of adenosine 3',5'-cyclicmonophosphate (cAMP) to reduce elevated levels of cytosolicCa2+ concentration([Ca2+]i)in pancreatic -cells.[Ca2+]iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single-cells by fura 2 and autofluorescence microfluorometry. Sustained[Ca2+]ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activatorforskolin (10 µM), and an incretin glucagon-likepeptide-1-(7-36) amide (109 M), as well as byglucose (16.7 mM). The[Ca2+]i-reducingeffects of cAMP were greater at elevated glucose (8.3-16.7 mM)than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA),H-89, counteracted[Ca2+]i-reducingeffects of cAMP but not those of glucose. Okadaic acid, a phosphataseinhibitor, at 10-100 nM also reduced sustained [Ca2+]ielevation in a concentration-dependent manner. Glucose, but not DBcAMP,increased NAD(P)H in -cells.[Ca2+]i-reducingeffects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitorof the endoplasmic reticulum (ER)Ca2+ pump. In contrast,[Ca2+]i-reducingeffects of cAMP were not altered by ryanodine, an ERCa2+-release inhibitor,Na+-free conditions, or diazoxide,an ATP-sensitive K+ channelopener. In conclusion, the cAMP-PKA pathway reduces[Ca2+]ielevation by sequestering Ca2+ inthapsigargin-sensitive stores. This process does not involve, but ispotentiated by, activation of -cell metabolism. Together with theknown[Ca2+]i-increasingaction of cAMP, our results reveal dual regulation of -cell[Ca2+]iby the cAMP-signaling pathway and by a physiological incretin.

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12.
Fast-twitch and slow-twitch rat skeletal muscles producedissimilar contractures with caffeine. We used digital imagingmicroscopy to monitor Ca2+ (withfluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes inCa2+ in individual fibers weremarkedly different from average responses of a population. All fibersshowed discrete, nonpropagated, local Ca2+ transients occurring randomlyin spots about one sarcomere apart. Caffeine increased localCa2+ transients and sarcomeremotion initially at 4 mM in soleus and 8 mM in extensor digitorumlongus (EDL; ~23°C). Ca2+release subsequently adapted or inactivated; this was surmounted byhigher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction inboth types of fiber. In EDL fibers, 16 mM caffeine moderately increasedlocal Ca2+ transients. In soleusfibers, 16 mM caffeine greatly increased Ca2+ release and producedpropagated waves of Ca2+(~1.5-2.5 µm/s). Ca2+waves in slow-twitch fibers reflect the caffeine-sensitive mechanism ofCa2+-inducedCa2+ release. Fast-twitch fiberspossibly lack this mechanism, which could account for their lowersensitivity to caffeine.

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13.
Published data suggest that the neuropeptide calcitonin gene-related peptide (CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca2+ concentration ([Ca2+]int) and membrane potential (Em) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC4(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca2+]int: a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC50 of 0.30 nM, and the maximal effect (initially 3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca2+ mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca2+ influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca2+ discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca2+ influx through L-type voltage-dependent Ca2+ channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular Em, with maximal effect at 20 nM and an EC50 of 0.30 nM. This effect was attenuated with charybdotoxin (–20%) or glyburide (glibenclamide; –80%), suggesting that Em hyperpolarization is induced by both Ca2+-activated and ATP-sensitive K+ channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells. osteoblastic cells; calcium; membrane potential; potassium channels; adenosine 3',5'-cyclic monophosphate  相似文献   

14.
This study investigated the effects of L-thyroxine-induced hyperthyroidism on Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca2+ pump (Ca2+-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca2+-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30–50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 (150%) Ca2+ pump isoform, unaltered levels of SERCA2 Ca2+ pump isoform, and lower levels of PLN (50%) and -, -, and -CaM kinase II (40 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca2+ uptake and Ca2+-stimulated ATPase activities compared with that from euthyroid control. The Vmax of Ca2+ uptake (in nmol Ca2+·mg SR protein–1·min–1: euthyroid, 818 ± 73; hyperthyroid, 1,649 ± 90) but not the apparent affinity of the Ca2+-ATPase for Ca2+ (euthyroid, 0.97 ± 0.02 µM, hyperthyroid, 1.09 ± 0.04 µM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca2+ uptake by soleus muscle SR was 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater (36%), and the time to peak force and relaxation time were significantly lower (30–40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca2+ pump. calmodulin kinase II; phospholamban ; calcium ion-adenosinetriphosphatase; sarcoplasmic reticulum  相似文献   

15.
The ryanodinereceptor mediates intracellularCa2+ mobilization in muscle andnerve, but its physiological role in nonexcitable cells is less welldefined. Like adenosine 3',5'-cyclic monophosphate andinositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 µM) andADP (1-25 µM) produced a concentration-dependent rise incytosolic Ca2+ in permeabilizedrat parotid acinar cells. Adenosine and AMP were less effective.Ryanodine markedly depressed theCa2+-mobilizing action of theadenine nucleotides and forskolin in permeabilized cells and waslikewise effective in depressing the action of forskolin in intactcells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced bycalmodulin and depressed by W-7, a calmodulin inhibitor. Afluorescently labeled ligand,4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression anddistribution of ryanodine receptors. In addition, ryanodine receptorexpression was detected in rat parotid cells with a sequence highlyhomologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of aryanodine-sensitive intracellularCa2+ store in rat parotid cellsthat shares many of the characteristics of stores in muscle and nerveand may mediate Ca2+-inducedCa2+ release or a modified form ofthis process.

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16.
We tested the effectof H-89, a protein kinase A (PKA) inhibitor, on the intracellulartransit of the regulated secretory proteins in rat lacrimal glands. Weshow that H-89, by itself, induces the secretion of newly synthesizedproteins trafficking in its presence but not of proteins already storedin the mature secretory granules. This secretion does not depend on thepresence of extracellular Ca2+. The proteins released areidentical to those secreted after cholinergic stimulation or under theaction of the ionophore A-23187, but the secretion level is ~40%lower. The effect of H-89 seems to be due to PKA inhibition becauseother protein kinase inhibitors (calphostin C, chelerythrine, H-85) donot induce secretion. We further show that H-89 does not modify therate of glycoprotein galactosylation but induces the secretion of newlygalactosylated glycoproteins. Finally, we used a "20°C block"procedure to show that H-89 affects a trans-Golgi network (TGN)or post-TGN step of the secretory pathway. Our results demonstratethat, in lacrimal cells, H-89 affects the intracellular trafficking ofsecretory proteins, suggesting a role for PKA in this process.

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17.
The mechanisms by which nitric oxide (NO) relaxes smooth muscles are unclear. The NO donor sodium nitroprusside (SNP) has been reported to increase the Ca2+ release frequency (Ca2+ sparks) through ryanodine receptors (RyRs) and activate spontaneous transient outward currents (STOCs), resulting in smooth muscle relaxation. Our findings that caffeine relaxes and hyperpolarizes murine gastric fundus smooth muscles and increases phospholamban (PLB) phosphorylation by Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) suggest that PLB phosphorylation by CaM kinase II participates in smooth muscle relaxation by increasing sarcoplasmic reticulum (SR) Ca2+ uptake and the frequencies of SR Ca2+ release events and STOCs. Thus, in the present study, we investigated the roles of CaM kinase II and PLB in SNP-induced relaxation of murine gastric fundus smooth muscles. SNP hyperpolarized and relaxed gastric fundus circular smooth muscles and activated CaM kinase II. SNP-induced CaM kinase II activation was prevented by KN-93. Ryanodine, tetracaine, 2-aminoethoxydiphenylborate, and cyclopiazonic acid inhibited SNP-induced fundus smooth muscle relaxation and CaM kinase II activation. The Ca2+-activated K+ channel blockers iberiotoxin and apamin inhibited SNP-induced hyperpolarization and relaxation. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one inhibited SNP-induced relaxation and CaM kinase II activation. The membrane-permeable cGMP analog 8-bromo-cGMP relaxed gastric fundus smooth muscles and activated CaM kinase II. SNP increased phosphorylation of PLB at Ser16 and Thr17. Thr17 phosphorylation of PLB was inhibited by cyclopiazonic acid and KN-93. Ser16 and Thr17 phosphorylation of PLB was sensitive to 1H-[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one. These results demonstrate a novel pathway linking the NO-soluble guanylyl cyclase-cGMP pathway, SR Ca2+ release, PLB, and CaM kinase II to relaxation in gastric fundus smooth muscles. calcium signaling; nitric oxide; sodium nitroprusside; calmodulin  相似文献   

18.
Ishimaru  Tadashi 《Chemical senses》1992,17(3):261-272
Reports are conflicting as to whether the presence of Ca2+ onthe ciliated surface of the olfactory mucosa suppresses or potentiatesthe response of receptor cells to odorants. To resolve thisissue, electro-olfactograms (EOGs) were recorded from the isolatedolfactory mucosae of the frog while its ciliated surface wasperfused with saline solutions containing differing concentrationsof Ca2+. A decrease in Ca2+ concentration augmented the EOGamplitude, and the magnitude of the augmentation increased asthe Ca2+ concentration decreased progressively. The slow forskolin-inducedchange in potential likewise increased in amplitude with theremoval of Ca2+ from the perfusate. Desensitization of EOG duringthe prolonged administration of odorants developed similarly,irrespective of the concentration of Ca2+ on the ciliated surface.These observations are consistent with findings of patch-clampand biochemical experiments. The augmentation of EOG at lowCa2+ concentrations appeared to result from either an increasein activity of adenylate cyclase or an increase in responsivenessof the channels activated by adenosine 3',5'-cyclic monophosphate,but not from an increased sensitivity of the receptor molecules.  相似文献   

19.
To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca2+ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca2+ concentration ([Ca2+]r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca2+]r and lower caffeine EC50 than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca2+]r in RyR3-expressing cells, studies of [3H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca2+]r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca2+]r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca2+]r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca2+]r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers  相似文献   

20.
Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca2+ waves that rely on Ca2+ release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca2+ waves in ICC. Intracellular [Ca2+] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca2+ waves in ICC and this was associated with a significant decrease in the ER Ca2+ load, measured with 10 mM caffeine responses. Ca2+ waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1 mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP3Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca2+ wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca2+ load sufficient to inactivate IP3Rs but not RyRs.  相似文献   

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