Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.     

Role of the inositol 1,4,5-trisphosphate receptor in Ca(2+) feedback inhibition of calcium release-activated calcium current (I(crac)).

We examined the activation and regulation of calcium release-activated calcium current (I(crac)) in RBL-1 cells in response to various Ca(2+) store-depleting agents. With [Ca(2+)](i) strongly buffered to 100 nM, I(crac) was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP(3)), and two metabolically stable IP(3) receptor agonists, adenophostin A and L-alpha-glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP(2)). With minimal [Ca(2+)](i) buffering, with [Ca(2+)](i) free to fluctuate I(crac) was activated by ionomycin, thapsigargin, and by the potent IP(3) receptor agonist, adenophostin A, but not by GPIP(2) or IP(3) itself. Likewise, when [Ca(2+)](i) was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP(2) and IP(3) did not activate detectable I(crac). However, with minimal [Ca(2+)](i) buffering, or with [Ca(2+)](i) buffered to 500 nM, GPIP(2) was able to fully activate detectable I(crac) if uptake of Ca(2+) intracellular stores was first inhibited. Our findings suggest that when IP(3) activates the IP(3) receptor, the resulting influx of Ca(2+) quickly inactivates the receptor, and Ca(2+) is re-accumulated at sites that regulate I(crac). Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca(2+) pools following activation by different IP(3) receptor agonists indicates that the critical regulatory pool of Ca(2+) may be very small in comparison to the total IP(3)-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP(3) receptors located on discrete IP(3)-sensitive Ca(2+) pools in calcium feedback regulation of I(crac) and capacitative calcium entry.     

Release of Ca(2+) from intracellular stores and entry of extracellular Ca(2+) are involved in sea squirt sperm activation.

A rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is required to activate sperm of all organisms studied. Such elevation of [Ca(2+)](i) can occur either by influx of extracellular Ca(2+) or by release of Ca(2+) from intracellular stores. We have examined these sources of Ca(2+) in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca(2+)-sensitive dye fura-2 to monitor [Ca(2+)](i) by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca(2+)], as well as by the presence of high [K(+)](o) or the Ca channel blockers pimozide, penfluridol, or Ni(2+), but not nifedipine or Co(2+). The accompanying rise in [Ca(2+)](i) was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca(2+) entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca(2+)](i) increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca(2+)](i) could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca(2+) from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP(3)) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca(2+) and that thimerosal stimulates release of Ca(2+) from IP(3)-sensitive intracellular stores.     

Beta-adrenergic potentiation of endoplasmic reticulum Ca(2+) release in brown fat cells

We find that the adrenergic agonist isoproterenol increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat brown adipocytes. At the concentration used (10 microM), isoproterenol-induced Ca(2+) responses were sensitive to block by either alpha(1)- or beta-adrenergic antagonists, suggesting an interaction between these receptor subtypes. Despite reliance on beta-adrenoceptor activation, the Ca(2+) response was not due solely to increases in cAMP because, administered alone, the selective beta(3)-adrenergic agonist BRL-37344 or forskolin did not increase [Ca(2+)](i). However, increased cAMP elicited vigorous [Ca(2+)](i) increases in the presence of barely active concentrations of the alpha-adrenergic agonist phenylephrine or the P2Y receptor agonist UTP. Consistent with isoproterenol recruiting only inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores, endoplasmic reticulum store depletion by thapsigargin blocked isoproterenol-induced Ca(2+) increases, but removal of external Ca(2+) did not. These results argue that increases in cAMP sensitize the IP(3)-mediated Ca(2+) release system in brown adipocytes.     

Intracellular Ca2+ regulation in rat motoneurons during development

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.     

Receptor-induced Ca(2+) signaling in cultured myenteric neurons

We studied the effect of excitatory neurotransmitters (10(-5) M) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of cultured myenteric neurons. ACh evoked a response in 48.6% of the neurons. This response consisted of a fast and a slow component, respectively mediated by nicotinic and muscarinic receptors, as revealed by specific agonists and antagonists. Substance P evoked a [Ca(2+)](i) rise in 68.2% of the neurons, which was highly dependent on Ca(2+) release from intracellular stores, since after thapsigargin (5 microM) pretreatment only 8% responded. The responses to serotonin, present in 90.7%, were completely blocked by ondansetron (10(-5) M), a 5-HT(3) receptor antagonist. Specific agonists of other serotonin receptors were not able to induce a [Ca(2+)](i) rise. Removing extracellular Ca(2+) abolished all serotonin and fast ACh responses, whereas substance P and slow ACh responses were more persistent. We conclude that ACh-induced signaling involves both nicotinic and muscarinic receptors responsible for a fast and a more delayed component, respectively. Substance P-induced signaling requires functional intracellular Ca(2+) stores, and the 5-HT(3) receptor mediates the serotonin-induced Ca(2+) signaling in cultured myenteric neurons.     

Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ influx through TRPC3

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca(2+)](i) increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca(2+)](i) in rat cultured spinal astrocytes using a Ca(2+) imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca(2+)](i) in a dose-dependent manner. The SP-induced increase in [Ca(2+)](i) was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca(2+)](i) was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP(3)) receptor inhibitor). In the absence of extracellular Ca(2+), GR73632 induced only a transient increase in [Ca(2+)](i). In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca(2+) release from intracellular Ca(2+) stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca(2+). RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca(2+)](i). These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca(2+) release from IP(3-)sensitive intracellular Ca(2+) stores, which is positively modulated by PKA, and subsequent Ca(2+) influx through TRPC3, which is negatively regulated by PKC.     

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

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

Novel effects of propranolol. Release of internal Ca(2+) followed by activation of capacitative Ca(2+) entry in Madin Darby canine kidney cells

The effect of propranolol on Ca(2+) signalling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Propranolol increased cytosolic free Ca(2+) levels ([Ca(2+)](i)) in a concentration-dependent manner between 0.1 and 1 mM. The response was partly inhibited by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with 0.2 mM propranolol partly inhibited the [Ca(2+)](i) rise induced by 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with thapsigargin abolished propranolol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 0.2 mM propranolol in Ca(2+)-free medium. Propranolol (0.2 mM) inhibited 25% of thapsigargin-induced capacitative Ca(2+) entry. Suppression of 1,4,5-trisphosphate (IP(3)) formation by 2 microM U73122, a phospholipase C inhibitor, did not alter 0.2 mM propranolol-induced internal Ca(2+) release. Propranolol (1 mM) also increased [Ca(2+)](i) in human neutrophils. Collectively, we have found that 0.2 mM propranolol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from thapsigargin-sensitive Ca(2+) stores in an IP(3)-independent manner, followed by Ca(2+) influx from external space. Independently, propranolol was able to inhibit thapsigargin-induced capacitative Ca(2+) entry.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

Developmental changes in capacitative Ca(2+) entry in mouse mammary epithelial cells

Developmental changes in capacitative Ca(2+) entry and Ca(2+) release from intracellular stores were measured using fura-2 fluorescence method during the pregnancy period (day 3-;18) in mouse mammary epithelial cells. Ca(2+) release was identified with the transient intracellular Ca(2+) ([Ca(2+)](i)) increase induced by thapsigargin addition in a Ca(2+)-free solution. Capacitative Ca(2+) entry was measured by the transient [Ca(2+)](i) increase induced by re-addition of extracellular Ca(2+) after depletion of Ca(2+) stores by thapsigargin. The capacitative Ca(2+) entry was greatest at the early stage of pregnancy (i.e. day 3 of pregnancy) and decreased as pregnancy progressed, while Ca(2+) release remained unchanged throughout the developmental stages. These findings indicate that in contrast to Ca(2+) release, a close correlation exists between capacitative Ca(2+) entry and pregnancy-induced development in mammary epithelial cells.     

Luminal Ca(2+) and the activity of sarcoplasmic reticulum Ca(2+) pumps modulate histamine-induced all-or-none Ca(2+) release in smooth muscle cells

We have studied histamine (HA)-evoked intracellular Ca(2+) release in single, freshly isolated myocytes from the guinea pig urinary bladder. Short applications of histamine (5 s) produced a thapsigargin (TG)-sensitive transient increase in intracellular calcium concentration ([Ca(2+)](i)). It was established that histamine and caffeine (Caff) released Ca(2+) from the same intracellular stores in these cells. Reducing the Ca(2+) content of internal stores by incubating cells with U-73343 or cyclopiazonic acid (CPA) inhibited the histamine-evoked Ca(2+) release in 69% and 60% of cells, respectively. Under these conditions, all cells released Ca(2+) in response to either caffeine or acetylcholine (ACh). However, decreasing internal Ca(2+) stores by removing external Ca(2+) inhibited histamine-induced Ca(2+) mobilization in only 22% of cells. A similar small fraction of cells was inhibited when sarcoplasmic reticulum (SR) Ca(2+) pumps were quickly blocked to avoid a significant reduction of luminal Ca(2+). In conclusion, lowering the luminal Ca(2+) content in combination with an impairment of the SR Ca(2+) pump activity significantly diminishes the ability of histamine to evoke an all-or-none intracellular Ca(2+) release.     

Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca(2+)](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor, caused a rapid rise in [Ca(2+)](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca(2+) stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca(2+)](i). However, the transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca(2+)](i) required IP(3) formation and binding to its specific receptors in Ca(2+) stores. Further studies were performed to investigate whether the effect of Cch on Ca(2+) entry into FRT cells was via L-type voltage-dependent Ca(2+) channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca(2+) channel blocker, decreased Cch-induced increase on [Ca(2+)](i), while Bay K-8644, an L-type Ca(2+) channel agonist, slightly increased [Ca(2+)](i) in FRT cells. These data indicate that Ca(2+) entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca(2+)](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca(2+) mobilization in neoplastic FRT cells.     

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。     

Hypotonic stress-induced dual Ca(2+) responses in bovine aortic endothelial cells

We have investigated the effects of hypotonic stress on intracellular calcium concentration ([Ca(2+)](i)) in bovine aortic endothelial cells. Reducing extracellular osmolarity by 5% to 40% elicited a steep Ca(2+) transient both in normal Krebs and Ca(2+)-free solutions. The hypotonic stress-induced Ca(2+) transient was inhibited by phospholipase C inhibitors (neomycin and U-73122), a P(2)-receptor antagonist (suramin), and an ATP-hydrolyzing enzyme (apyrase), suggesting that the hypotonic stress-induced Ca(2+) transient is mediated by ATP. A luciferin-luciferase assay confirmed that 40% hypotonic stress released 91.0 amol/cell of ATP in 10 min. When the hypotonic stress-induced fast Ca(2+) transient was inhibited by neomycin, suramin, or apyrase, a gradual [Ca(2+)](i) increase was observed instead. This hypotonic stress-induced gradual [Ca(2+)](i) increase was inhibited by a phospholipase A(2) inhibitor, 4-bromophenacyl bromide. Furthermore, exogenously applied arachidonic acid induced a gradual [Ca(2+)](i) increase with an ED(50) of 13.3 microM. These observations indicate that hypotonic stress induces a dual Ca(2+) response in bovine aortic endothelial cells, i.e., an ATP-mediated fast Ca(2+) transient and an arachidonic acid-mediated gradual Ca(2+) increase, the former being the predominant response in normal conditions.     

Increased Ca(2+) sparks and sarcoplasmic reticulum Ca(2+) stores potentially determine the spontaneous activity of pulmonary vein cardiomyocytes

Pulmonary veins (PVs) contain cardiomyocytes with spontaneous activity that may be responsible for PV arrhythmia. Abnormal Ca(2+) regulation is known to contribute to PV arrhythmogenesis. The purpose of this study was to investigate whether PV cardiomyocytes with spontaneous activity have different intracellular Ca(2+) ([Ca(2+)](i)) transients, Ca(2+) sparks and responses to isoproterenol and ryanodine receptor modulators (magnesium and FK506) than do PV cardiomyocytes without spontaneous activity and left atrial (LA) cardiomyocytes. Through fluorescence and confocal microscopy, we evaluated the [Ca(2+)](i) transients and Ca(2+) sparks in isolated rabbit PV and LA cardiomyocytes. PV cardiomyocytes with spontaneous activity had larger [Ca(2+)](i) transients and sarcoplasmic reticulum (SR) Ca(2+) stores than PV cardiomyocytes without spontaneous activity or LA cardiomyocytes. PV cardiomyocytes with spontaneous activity also had a higher incidence and frequency of Ca(2+) sparks, and had Ca(2+) sparks with larger amplitudes than other cardiomyocytes. Magnesium (5.4 mM) reduced the [Ca(2+)](i) transient amplitude and beating rate in PV cardiomyocytes with spontaneous activity. However, in contrast with other cardiomyocytes, low doses (1.8 mM) of magnesium did not reduce the [Ca(2+)](i) transients amplitude in PV cardiomyocytes with spontaneous activity. FK506 (1 muM) diminished the SR Ca(2+) stores in PV cardiomyocytes with spontaneous activity to a lesser extent than that in other cardiomyocytes. Isoproterenol (10 nM) increased the [Ca(2+)](i) transient amplitude to a lesser extent in LA cardiomyocytes than in PV cardiomyocytes with or without spontaneous activity. In conclusion, our results suggest that enhanced [Ca(2+)](i) transients, increased Ca(2+) sparks and SR Ca(2+) stores may contribute to the spontaneous activity of PV cardiomyocytes.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.