Septal interstitial cells of Cajal conduct pacemaker activity to excite muscle bundles in human jejunum

BACKGROUND & AIMS: Like the heart, intestinal smooth muscles exhibit electrical rhythmicity, which originates in pacemaker cells surrounding the myenteric plexus, called interstitial cells of Cajal (ICC-MY). In large mammals, ICC also line septa (ICC-SEP) between circular muscle (CM) bundles, suggesting they might be necessary for activating muscle bundles. It is important to determine their functional significance, because a loss of ICC in humans is associated with disordered motility. Our aims were therefore to determine the role of ICC-SEP in activating the thick CM in the human jejunum. METHODS: The mucosa and submucosa were removed and muscle strips were cut and pinned in cross-section so that the ICC-MY and ICC-SEP networks and the CM could be readily visualized. The ICC networks and CM were loaded with the Ca(2+) indicator fluo-4, and pacemaker and muscle activity was recorded at 36.5 +/- 0.5( degrees )C. RESULTS: Ca(2+) imaging revealed that pacemaker activity in human ICC-MY can entrain ICC-SEP to excite CM bundles. Unlike the heart, pacemaker activity in ICC-MY varied in amplitude, propagation distance, and direction, leading to a sporadic activation of ICC-SEP. CONCLUSIONS: ICC-SEP form a crucial conduction pathway for spreading excitation deep into muscle bundles of the human jejunum, necessary for motor patterns underlying mixing. A loss of these cells could severely affect motor activity.     

Cajal 间质细胞与胃肠起搏

近年的研究发现,Cajal间质细胞(ICC)是胃肠起博细胞。ICC呈星状,有长的突起。均表达c-kit,ICC之间、ICC与周围平滑肌形成缝隙连接,可自发产生起博电位。ICC起博的机制可能是:ICC自发产生的单元电位总和达阈值,激活电压依赖的Ca2 可通透的离子通道,形成起博电位的初始部分;Ca2 内流,激活对细胞内Ca2 敏感的酶,使IP3生成增加;从而增高IP3的浓度,引起Ca2 从内源性Ca2 库瞬间释放,使细胞内Ca2 浓度上升,活化细胞膜上Ca2 活化的Cl-通道,细胞膜去极化产生平台部分;Ca2 的进入使局部Ca2 浓度上升,通道失活,起搏电位终止。     

Inositol 1,4,5-trisphosphate receptors and pacemaker rhythms

Intracellular Ca(2+) plays an important role in the control of the heart rate through the interaction between Ca(2+) release by ryanodine receptors in the sarcoplasmic reticulum (SR) and the extrusion of Ca(2+) by the sodium-calcium exchanger which generates an inward current. A second type of SR Ca(2+) release channel, the inositol 1,4,5-trisphosphate receptor (IP(3)R), can release Ca(2+) from SR stores in many cell types, including cardiac myocytes. However, it is still uncertain whether IP(3)Rs play any functional role in regulating the heart rate. Accumulated evidence shows that IP(3) and IP(3)R are involved in rhythm control in non-cardiac pacemaker tissues and in the embryonic heart. In this review we focus on intracellular Ca(2+) oscillations generated by Ca(2+) release from IP(3)R that initiates membrane depolarization and provides a common mechanism producing spontaneous activity in a range of cells with pacemaker function. Emerging new evidence also suggests that IP(3)/IP(3)Rs play a functional role in normal and diseased hearts and in cardiac rhythm control. Several membrane currents, including a store-operated Ca(2+) current, might be activated by Ca(2+) release from IP(3)Rs. IP(3)/IP(3)R may thus add another dimension to the complex regulation of heart rate.     

Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells

Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca(2+) signals is uncharacterized. We examined Ca(2+)-dependent insulin signaling in human beta cells. Two hundred nanomolar insulin elevated [Ca(2+)](c) to 284 +/- 27 nM above baseline in approximately 30% of Fura-4F-loaded cells. Insulin evoked multiple Ca(2+) signal waveforms, 60% of which included oscillations. Although the amplitude of Ca(2+) signals was dose-dependent between 0.002 and 2,000 nM, the percentage of cells responding was highest at 0.2 nM insulin, suggesting the interaction of stimulatory and inhibitory pathways. Ca(2+)-free solutions did not affect the initiation of insulin-stimulated Ca(2+) signals, but abolished the second phase of plateaus/oscillations. Likewise, inositol 1,4,5-trisphosphate (IP(3)) receptor antagonists xestospongin C and caffeine selectively blocked the second phase, but not the initiation of insulin signaling. Thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ) blocked insulin signaling, implicating sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA)-containing Ca(2+) stores. Insulin-stimulated Ca(2+) signals were insensitive to ryanodine. Injection of the CD38-derived Ca(2+) mobilizing metabolite, nicotinic acid-adenine dinucleotide phosphate (NAADP), at nanomolar concentrations, evoked oscillatory Ca(2+) signals that could be initiated in the presence of ryanodine, xestospongin C, and Ca(2+)-free solutions. Desensitizing concentrations of NAADP abolished insulin-stimulated Ca(2+) signals. Insulin-stimulated Ca(2+) signals led to a Ca(2+)-dependent increase in cellular insulin contents, but not secretion. These data reveal the complexity of insulin signal transduction and function in human beta cells and demonstrate functional NAADP-sensitive Ca(2+) stores in a human primary cultured cell type.     

Transfected cGMP-dependent protein kinase suppresses calcium transients by inhibition of inositol 1,4,5-trisphosphate production.

cGMP is a key regulatory molecule in visual transduction, integration of neuronal response to excitatory neurotransmitters, relaxation of smooth muscle, intestinal secretion of water and salt, and reabsorption of sodium and water in the distal tubules of the nephron. Some of these cellular functions are associated with the activation of cGMP kinase and a decrease in cytosolic calcium levels ([Ca2+]i). The mechanism by which cGMP kinase lowers [Ca2+]i is controversial. We have used CHO cells stably transfected with cGMP kinase to test several of the proposed [Ca2+]i-lowering mechanisms. Thrombin induces a calcium transient in wild-type and cGMP kinase-expressing CHO cells by releasing calcium from intracellular stores. Preincubation of wild-type cells with 8-bromo-cGMP had no effect on the calcium transient, whereas 8-bromo-cGMP prevented the thrombin-stimulated calcium transient in cGMP kinase-expressing CHO cells. In both cell types 8-bromo-cGMP had no effect on [Ca2+]i transients induced by replacing extracellular sodium by tetramethylammonium, ruling out an effect of cGMP kinase on Ca(2+)-ATPases. However, cGMP kinase activation effectively suppressed thrombin-induced stimulation of inositol 1,4,5-trisphosphate production. These results show that cGMP kinase lowers [Ca2+]i by interfering with the inositol 1,4,5-trisphosphate synthesis.     

The role of cyclic-ADP-ribose-signaling pathway in oxytocin-induced Ca2+ transients in human myometrium cells

Human myometrial contraction plays a fundamental role in labor. Dysfunction of uterine contraction is an important cause of labor progression failure. Although the mechanisms controlling uterine contraction are not completely understood, intracellular Ca2+ mobilization plays an important role during uterine contraction. Several mechanisms of intracellular Ca2+ mobilization are present in smooth muscle, but in the human uterus, only 1,4,5-trisphosphate-induced Ca2+ release has been studied extensively. Ryanodine receptor channels are present in myometrium. We determined the role of the cyclic ADP-ribose (cADPR)-signaling pathway in oxytocin-induced intracellular Ca2+ [(Ca2+)i] transients in human myometrial cells. We found that oxytocin-induced Ca2+ transient is dependent on several sources of Ca2+, including extracellular Ca2+ and intracellular Ca2+ stores. In addition, we found that both the 1,4,5-trisphosphate- and the cADPR-induced Ca2+ releasing systems are important for the induction of [Ca2+]i transients by oxytocin in human myometrial cells. Furthermore, we investigated TNFalpha regulation of oxytocin-induced [Ca2+]i transients, CD38 cyclase activity, and CD38 expression in human myometrial cells. We found that oxytocin-induced [Ca2+]i transients were significantly increased by 50 ng/ml TNF. Similarly, CD38 mRNA levels, CD38 expression, and cyclase activity were increased by TNFalpha, thus increasing cADPR levels. We propose that a complex interaction between multiple signaling pathways is important for the development of intracellular Ca2+ transients induced by oxytocin and that TNFalpha may contribute for the myometrium preparation for labor by regulating the cADPR-signaling pathway. The observation that the cADPR-signaling pathway is important for the development of intracellular Ca2+ transients in human myometrial cells raises the possibility that this signaling pathway could serve as a target for the development of new therapeutic strategies for abnormal myometrial contraction observed during pregnancy.     

The ERK cascade,a new pathway involved in the activation of store-mediated calcium entry in human platelets

Store-mediated Ca(2+) entry (SMCE) is the main pathway for Ca(2+) influx in platelets and other nonexcitable cells, yet how depletion of the intracellular Ca(2+) stores leads to the activation of Ca(2+) entry across the plasma membrane remains unclear. Recent work in platelets favors a secretionlike conformational coupling mechanism involving the Ca(2+)-permeable channel protein, Trp1, in the plasma membrane and the type-II inositol 1,4,5-trisphosphate receptor in the membrane of the Ca(2+) store, which is located in the endoplasmic reticulum. Extracellular signal-regulated kinases (ERKs) are common participants in a broad variety of signal transduction pathways in human platelets, and inactivation of the ERK cascade has been shown to reduce Ca(2+) entry stimulated by thapsigargin or thrombin. The role of ERK in SMCE into human platelets was found to be independent of the cytoskeleton and a downstream effector of the small guanosine-triphosphate-binding protein Ras.     

Lipid rafts establish calcium waves in hepatocytes

BACKGROUND & AIMS: Polarity is critical for hepatocyte function. Ca(2+) waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca(2+) waves depends on lipid rafts. METHODS: Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy. RESULTS: Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD. CONCLUSIONS: Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.     

Sinoatrial node pacemaker activity requires Ca(2+)/calmodulin-dependent protein kinase II activation

Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 micromol/L), or by KN-93 (0.1 to 3.0 micromol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 micromol/L AIP and 3 micromol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca(2+) current (I(Ca, L)), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 micromol/L), but not its inactive analog, KN-92, decreased I:(Ca, L) amplitude from 12+/-2 to 6+/-1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I(Ca, L) from inactivation. Similar results were observed using the fast Ca(2+) chelator BAPTA, whereas the slow Ca(2+) chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca(2+) transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I(Ca, L) inactivation and reactivation, and local Ca(2+) is critically involved in these processes.     

Membrane hyperpolarization is not required for sustained muscarinic agonist-induced increases in intracellular Ca2+ in arteriolar endothelial cells

OBJECTIVE: Hyperpolarization modulates Ca2+ influx during agonist stimulation in many endothelial cells, but the effects of hyperpolarization on Ca2+ influx in freshly isolated arteriolar endothelial cells are unknown. Therefore, the purpose of the present study was to characterize agonist-induced Ca2+ transients in freshly isolated arteriolar endothelial cells and to test the hypothesis that membrane hyperpolarization augments agonist-induced Ca2+ influx into these cells. METHODS: Arterioles were removed from hamster cremaster muscles and arteriolar endothelial cells were enzymatically isolated. Endothelial cells were loaded with Fura 2-AM and the Fura 2 ratio measured photometrically as an index of intracellular Ca2+. The cells were then stimulated with the muscarinic, cholinergic agonist, methacholine, and the resulting Ca2+ transients were measured. RESULTS: Methacholine (1 microM) increased the endothelial cell Fura 2 ratio from a baseline of 0.81 +/- 0.02 to an initial peak of 1.17 +/- 0.05 (n = 17) followed by a sustained plateau of 1.12 +/- 0.07. The plateau phase of the Ca2+ transient was inhibited by removal of extracellular Ca2+ (n = 12, p < .05), or the nonselective cation channel blockers Gd3+ (30 microM; n = 7, p < .05) or La3+ (50 microM; n = 7, p < .05) without significant effect on the baseline or peak (p > .05). The initial peak of methacholine-induced Ca2+ transients was inhibited by the IP3-receptor antagonist xestospongin D (10 microM, n = 5, p < .05). The methacholine-induced Ca2+ transients were accompanied by endothelial cell hyperpolarization of approximately 14-18 mV, as assessed by experiments using the potentiometric dye, di-8-ANEPPS as well as by patch-clamp experiments. However, inhibition of hyperpolarization by blockade of Ca2+-activated K+ channels with charybdotoxin (100 nM) and apamin (100 nM) (n = 5), or exposure of endothelial cells to 80 or 145 mM KCl (both n = 7) had no effect on the plateau phase of methacholine-induced Ca2+ transients (p > .05). CONCLUSIONS: Freshly isolated arteriolar endothelial cells display agonist-induced Ca2+ transients. For the muscarinic agonist, methacholine, these Ca2+ transients result from release of Ca2+ from intracellular stores through IP3 receptors, followed by sustained influx of extracellular Ca2+. While these changes in intracellular Ca2+ are associated with endothelial cell hyperpolarization, the methacholine-induced, sustained increase in intracellular Ca2+ appears to be independent from this change in membrane potential. These data suggest that arteriolar endothelial cells may possess a novel Ca2+ influx pathway, or that the relationship between intracellular Ca2+ and Ca2+ influx is more complex than that observed in other endothelial cells.     

Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis

BACKGROUND AND AIMS: Cholestasis is one of the principal manifestations of liver disease and often results from disorders involving bile duct epithelia rather than hepatocytes. A range of disorders affects biliary epithelia, and no unifying pathophysiologic event in these cells has been identified as the cause of cholestasis. Here we examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R)/Ca(2+) release channel in Ca(2+) signaling and ductular secretion in animal models of cholestasis and in patients with cholestatic disorders. METHODS: The expression and distribution of the InsP3R and related proteins were examined in rat cholangiocytes before and after bile duct ligation or treatment with endotoxin. Ca(2+) signaling was examined in isolated bile ducts from these animals, whereas ductular bicarbonate secretion was examined in isolated perfused livers. Confocal immunofluorescence was used to examine cholangiocyte InsP3R expression in human liver biopsy specimens. RESULTS: Expression of the InsP3R was selectively lost from biliary epithelia after bile duct ligation or endotoxin treatment. As a result, Ca(2+) signaling and Ca(2+)-mediated bicarbonate secretion were lost as well, although other components of the Ca(2+) signaling pathway and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated bicarbonate secretion both were preserved. Examination of human liver biopsy specimens showed that InsP3Rs also were lost from bile duct epithelia in a range of human cholestatic disorders, although InsP3R expression was intact in noncholestatic liver disease. CONCLUSIONS: InsP3-mediated Ca(2+) signaling in bile duct epithelia appears to be important for normal bile secretion in the liver, and loss of InsP3Rs may be a final common pathway for cholestasis.     

The type II inositol 1,4,5-trisphosphate receptor can trigger Ca2+ waves in rat hepatocytes

BACKGROUND & AIMS: Ca2+ regulates cell functions through signaling patterns such as Ca2+ oscillations and Ca2+ waves. The type I inositol 1,4,5-trisphosphate receptor is thought to support Ca2+ oscillations, whereas the type III inositol 1,4,5-trisphosphate receptor is thought to initiate Ca2+ waves. The role of the type II inositol 1,4,5-trisphosphate receptor is less clear, because it behaves like the type III inositol 1,4,5-trisphosphate receptor at the single-channel level but can support Ca2+ oscillations in intact cells. Because the type II inositol 1,4,5-trisphosphate receptor is the predominant isoform in liver, we examined whether this isoform can trigger Ca2+ waves in hepatocytes. METHODS: The expression and distribution of inositol 1,4,5-trisphosphate receptor isoforms was examined in rat liver by immunoblot and confocal immunofluorescence. The effects of inositol 1,4,5-trisphosphate on Ca2+ signaling were examined in isolated rat hepatocyte couplets by using flash photolysis and time-lapse confocal microscopy. RESULTS: The type II inositol 1,4,5-trisphosphate receptor was concentrated near the canalicular pole in hepatocytes, whereas the type I inositol 1,4,5-trisphosphate receptor was found elsewhere. Stimulation of hepatocytes with vasopressin or directly with inositol 1,4,5-trisphosphate induced Ca2+ waves that began in the canalicular region and then spread to the rest of the cell. Inositol 1,4,5-Trisphosphate-induced Ca2+ signals also increased more rapidly in the canalicular region. Hepatocytes did not express the ryanodine receptor, and cyclic adenosine diphosphate-ribose had no effect on Ca2+ signaling in these cells. CONCLUSIONS: The type II inositol 1,4,5-trisphosphate receptor establishes a pericanalicular trigger zone from which Ca2+ waves originate in hepatocytes.     

Sonic hedgehog signaling is decoded by calcium spike activity in the developing spinal cord

Evolutionarily conserved hedgehog proteins orchestrate the patterning of embryonic tissues, and dysfunctions in their signaling can lead to tumorigenesis. In vertebrates, Sonic hedgehog (Shh) is essential for nervous system development, but the mechanisms underlying its action remain unclear. Early electrical activity is another developmental cue important for proliferation, migration, and differentiation of neurons. Here we demonstrate the interplay between Shh signaling and Ca(2+) dynamics in the developing spinal cord. Ca(2+) imaging of embryonic spinal cells shows that Shh acutely increases Ca(2+) spike activity through activation of the Shh coreceptor Smoothened (Smo) in neurons. Smo recruits a heterotrimeric GTP-binding protein-dependent pathway and engages both intracellular Ca(2+) stores and Ca(2+) influx. The dynamics of this signaling are manifested in synchronous Ca(2+) spikes and inositol triphosphate transients apparent at the neuronal primary cilium. Interaction of Shh and electrical activity modulates neurotransmitter phenotype expression in spinal neurons. These results indicate that electrical activity and second-messenger signaling mediate Shh action in embryonic spinal neurons.     

Effect of endothelin-1 on cytosolic calcium ions in cultured human endothelial cells

The effects of endothelin-1 on cytosolic Ca2+ and inositol 1,4,5-triphosphate (IP3) levels were investigated in cultured, untreated human endothelial cells and in endothelial cells pretreated with anti-endothelin-1 serum for 24 h to exclude the effect of endogenous endothelin-1. Endothelin-1 was found to increase the intracellular Ca2+ level, either in the presence or absence of extracellular Ca2+, in endothelial cells pretreated with antiserum by the fura-2 fluorescence technique. IP3 levels immediately started to rise following endothelin-1 stimulation. Resting intracellular Ca2+ levels were significantly lower when the cells were pretreated with antiserum than without antiserum pretreatment. Following stimulation by endothelin-1, intracellular Ca2+ and IP3 levels in endothelial cells pretreated with antiserum increased significantly compared to those in untreated endothelial cells. Endothelin-1 also increased 45Ca influx from the extracellular space. These results suggest that endothelin-1 increases intracellular Ca2+ in endothelial cells through extracellular Ca2(+)-dependent mechanisms and by the release of Ca2+ from intracellular stores, this presumably being induced by IP3 formation.     

2APB- and JTV519(K201)-sensitive micro Ca waves in arrhythmogenic Purkinje cells that survive in infarcted canine heart

OBJECTIVES/BACKGROUND: Studies from several laboratories have implicated intracellular Ca(2+) dynamics in the modulation of electrical activity. We have reported that abnormal Ca(2+) wave activity is the underlying cause of afterdepolarization-induced electrical activity in subendocardial Purkinje cells that survive in the 48-hour infarcted canine heart. These cells form the focus of arrhythmias at this time postcoronary artery occlusion. METHODS: We studied the effects of agonists and antagonists on the abnormal Ca(2+) release activity of Purkinje cell aggregates dispersed from the subendocardium 48 hours postcoronary artery occlusion (IZPCs). Studies were completed using epifluorescent microscopy of Fluo-3 loaded Purkinje cells. RESULTS: Similar to our previous report, highly frequent traveling micro Ca(2+) transients (muCaiTs) and cell-wide Ca(2+) waves were seen in IZPCs in the absence of any drug. Isoproterenol (ISO) increased muCaiTs and cell-wide Ca(2+) waves in Purkinje cells dispersed from the normal heart (NZPCs). In IZPCs, ISO increased cell-wide wave frequency but had no effect on the already highly frequent micro Ca(2+) wave transient activity, suggesting that ISO lowers the threshold of cell-wide generators responding to micro Ca(2+) transients. Drugs that block inward sodium or calcium currents (verapamil, tetrodotoxin) had no effect on Ca(2+) activity in Purkinje cells. Antagonists of intracellular Ca(2+) release channels [ryanodine, JTV519(K201)] greatly suppressed spontaneous Ca(2+) release events in IZPCs. 2APB, an agent that blocks IP(3) receptors, greatly reduced the frequency of Ca(2+) events in IZPCs. CONCLUSIONS: In arrhythmogenic Purkinje cells that survive in the infarcted heart, agents that block or inhibit intracellular Ca(2+) release channel activity reduced Ca(2+) waves and could be antiarrhythmic.     

Heart failure enhanced pulmonary vein arrhythmogenesis and dysregulated sodium and calcium homeostasis with increased calcium sparks

Late sodium currents and intracellular Ca(2+) (Ca(2+) (i)) dynamics play an important role in arrhythmogenesis of pulmonary vein (PV) and heart failure (HF). It is not clear whether HF enhances PV arrhythmogenesis through modulation of Ca(2+) homeostasis and increased late sodium currents. The aim of this study was to investigate the sodium and calcium homeostasis in PV cardiomyocytes with HF. METHODS AND RESULTS: Whole-cell patch clamp was used to investigate the action potentials and ionic currents in isolated rabbit single PV cardiomyocytes with and without rapid pacing induced HF. The Ca(2+) (i) dynamics were evaluated through fluorescence and confocal microscopy. As compared to control PV cardiomyocytes (n = 18), HF PV cardiomyocytes (n = 13) had a higher incidence of delayed afterdepolarization (45% vs 13%, P < 0.05) and faster spontaneous activity (3.0 ± 0.2 vs 2.1 ± 0.2 Hz, P < 0.05). HF PV cardiomyocytes had increased late Na(+) currents, Na(+) /Ca(2+) exchanger currents, and transient inward currents, but had decreased Na(+) currents or L-type calcium currents. HF PV cardiomyocytes with pacemaker activity had larger Ca(2+) (i) transients (R410/485, 0.18 ± 0.04 vs 0.11 ± 0.02, P < 0.05), and sarcoplasmic reticulum Ca(2+) stores. Moreover, HF PV cardiomyocytes with pacemaker activity (n = 18) had higher incidence (95% vs 70%, P < 0.05), frequency (7.8 ± 3.1 vs 2.3 ± 1.2 spark/mm/s, P < 0.05), amplitude (F/F(0) , 3.2 ± 0.8 vs 1.9 ± 0.5, P < 0.05), and longer decay time (65 ± 3 vs 48 ± 4 ms, P < 0.05) of Ca(2+) sparks than control PV cardiomyocytes with pacemaker activity (n = 18). CONCLUSIONS: Dysregulated sodium and calcium homeostasis, and enhanced calcium sparks promote arrhythmogenesis of PV cardiomyocytes in HF, which may play an important role in the development of atrial fibrillation.     

Nonuniform Ca2+ transients in arrhythmogenic Purkinje cells that survive in the infarcted canine heart

OBJECTIVE AND METHODS: In this study, we investigated whether Ca(2+) transients are altered in Purkinje cell aggregates dispersed from the subendocardium overlying the infarcted zone of the left ventricle (IZPCs) 48 h after coronary artery occlusion. To do so, we combined epifluorescent imaging with microelectrode recordings of IZPCs and normal canine Purkinje cell aggregates (NZPCs). RESULTS: NZPCs respond to an action potential (AP) by a small Ca(2+) transient at the cell surface immediately after the AP upstroke followed by a large [Ca(2+)] transient, which propagates to the cell core. In addition, focal Ca(2+) waves can originate spontaneously later during the AP or during the diastolic interval (Circ Res 2000;86:448-55) and then propagate throughout the aggregate as 'cell-wide Ca(2+) waves'. Electrically-evoked Ca(2+) transients in IZPCs arose significantly faster than those in NZPCs, and showed substantial spatiotemporal nonuniformity within an IZPC aggregate as well as between IZPC aggregates. IZPCs showed, hitherto undetected, low amplitude, micro Ca(2+) transients (extent 相似文献   

The phospholipase C-InsP3 pathway is involved in calcium mobilization induced by growth hormone in hepatocytes.

We investigated the effects of bovine GH (bGH) on Ca(2+) handling, phospholipase C (PLC) activation and inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation in hepatocytes. bGH generates oscillations in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in single male rat hepatocytes microinjected with the photoprotein aequorin. In the absence of extracellular Ca(2+) these transients persisted for more than 10 min indicating a requirement for intracellular Ca(2+). Treatment of the hepatocyte with the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U-73122 removed the oscillations. These results suggest bGH-induced oscillations are due to PLC activation and generation of Ins(1,4,5)P(3). We measured the mass of Ins(1,4,5)P(3) in freshly isolated hepatocyte suspensions in response to bGH, and vasopressin as a control. Both agonists rapidly increased the levels of Ins(1,4,5)P(3). This is the first study to indicate that early events in the signal transduction pathways mediated by GH in hepatocytes involve intracellular Ca(2+) mobilization via activation of a PI-PLC and subsequent Ins(1,4,5)P(3) production.     

Comparison of sarcoplasmic reticulum Ca2+-ATPase function in human,dog, rabbit,and mouse ventricular myocytes

It has been reported that sarcoplasmic reticulum (SR) Ca(2+) uptake is more rapid in rat than rabbit ventricular myocytes, but little information is available on the relative SR Ca(2+) uptake activity in others species, including humans. We induced Ca(2+) transients with a short caffeine pulse protocol (rapid solution switcher, 10 mM caffeine, 100 ms) in single ventricular myocytes voltage clamped (-80 mV) with pipettes containing 100 microM fluo-3 and nominal 0 Ca(2+), in 0 Na(+)(o)/0 Ca(2+)(o) solution to inhibit Na/Ca exchange. SR in non-paced human, dog, rabbit, and mouse ventricular myocytes could be readily loaded with Ca(2+) under our experimental conditions with a pipette [Ca(2+)] = 100 nM. Resting [Ca(2+)](i) was similar in four types of ventricular myocytes. Activation of the Ca(2+)-release channel with a 100-ms caffeine pulse produced a rise in [caffeine](i) to slightly above 2 mM, the threshold for caffeine activation of Ca(2+) release. This caused a similar initial rate of rise and peak [Ca(2+)](i) in the four types of ventricular myocytes. However, there were significant differences in the duration of the plateau (top 10%) [Ca(2+)](i) transients and the time constant of the [Ca(2+)](i) decline (reflecting activity of the SR Ca(2+)-ATPase), with values for human > dog > rabbit > mouse. In paced myocytes under physiologic conditions, SR Ca(2+) content was greater in mouse than in rabbit myocytes, while peak I(Ca,L) was smaller in mouse. These findings confirm substantial species difference in SR Ca(2+)-ATPase activity, and suggest that the smaller the animal and the more rapid the heart rate, greater the activity of the SR Ca(2+)-ATPase. In addition, it appears that substantial species differences exist in the degree of SR Ca(2+) loading and I(Ca,L) under physiologic conditions.     

Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells.

Calcium ions play a central role in stimulus-secretion coupling in pancreatic beta cells, and an elevation of cytosolic Ca2+ levels is necessary for insulin secretion. Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ stores in the beta cell by binding to specific receptors that are ligand-activated Ca2+ channels. The inositol trisphosphate receptors comprise a family of structurally related proteins with distinct but overlapping tissue distributions. Previous studies indicated that the predominant inositol trisphosphate receptor subtype expressed in rat pancreatic islets was the protein designated IP3R-3. We have confirmed the expression of IP3R-3 in pancreatic islets by immunohistocytochemistry and localized this protein to the secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells by immunogold electron microscopy. Secretory granules contain high levels of Ca2+, and the presence of IP3R-3 in the granule provides a mechanism for mobilizing granule Ca2+ stores in response to glucose and/or hormones. The release of Ca2+ from granule stores would increase the Ca2+ concentration in the surrounding cytoplasm and promote rapid exocytosis of granules, especially those granules in close proximity to the plasma membrane. The levels of IP3R-3 were increased in pancreatic islets of diabetic rats and rats that had been refed after a period of fasting. They were also increased in rat insulinoma RINm5F cells cultured in 25 mM glucose compared with cells cultured in 5 mM glucose. The localization of IP3R-3 to secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells suggests that granule Ca2+ stores actively participate in the secretory process and that their release is regulated by inositol 1,4,5-trisphosphate. The regulation of IP3R-3 levels by glucose, diabetes, and refeeding may allow the beta cell to adjust the insulin secretory response to changing physiological conditions.