The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent

Loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. However, the nature of the stimulus that activates the cell is debated. Candidate stimuli include wall shear stress, streaming potentials, and chemotransport. We have addressed the nature of the flow-derived cell stimulus by comparing variations in fluid transport with variations in wall shear stress, using nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production as a parameter of bone cell activation. Adult mouse long bone cell cultures were treated for 15min with or without pulsating fluid flow using the following regimes: Low PFF, mean flow rate 0.20 cm(3)/s, 3 Hz, shear stress 0.4+/-0.12 Pa; Medium PFF, 0.33 cm(3)/s, 5 Hz, 0.6+/-0.27 Pa; and High PFF, 0.63 cm(3)/s, 9Hz, 1.2+/-0.37 Pa. In some Low PFF experiments, 2.8% neutral dextran (mol. wt. 4.98x10(4)) was added to the flow medium to increase the viscosity, thereby increasing the wall shear stress 3-fold to a level similar of the High PFF stimulus, but without affecting streaming potentials or chemotransport. NO and PGE(2) production were stimulated by Low, Medium, and High PFF in a dose-dependent manner. Application of Low PFF using dextran-supplemented medium, enhanced both the NO and PGE(2) response by 3-fold, to a level mimicking the response to High PFF at normal viscosity. These results show that the production of NO and PGE(2) by bone cells can be enhanced in a dose-dependent manner by fluid flow of increasing wall shear stress. Therefore, the stimulus leading to NO and PGE(2) production is the flow-derived shear stress, and not streaming potentials or chemotransport.     

Osteoblastic cells have refractory periods for fluid-flow-induced intracellular calcium oscillations for short bouts of flow and display multiple low-magnitude oscillations during long-term flow

Partitioning a daily mechanical stimulus into discrete loading bouts enhances bone formation in rat tibiae (J. Bone Mineral Res. 15(8) (2000) 1596). We hypothesized that a refractory period exists in primary rat osteoblastic cells, during which fluid-flow-induced [Ca(2+)](i) oscillations are insensitive to additional short bouts (2 min) of fluid flow. Because the frequency of [Ca(2+)](i) oscillations is believed to be important for regulating cellular activity and long-term fluid flow alters gene expression in bone cells, we also hypothesized that long-term (15 min) oscillating fluid flow produces multiple [Ca(2+)](i) oscillations in osteoblastic cells. Primary osteoblastic cells from rat long bones were exposed to 2 min of oscillating fluid flow that produced shear stresses of 2 Pa at 2 Hz. After a rest period of 5, 30, 60, 300, 600, 900, 1800, or 2700 s, the cells were exposed to a second 2-min bout of flow. A 600 s rest period was required to recover the percentage of cells responding to fluid flow and a 900 s rest period was required to recover the [Ca(2+)](i) oscillation magnitude. The magnitude and shape of the two [Ca(2+)](i) oscillations were strikingly similar for individual cells after a 900 s rest period. During 15 min of continuous oscillating flow, some individual cells displayed between 1 and 9 oscillations subsequent to the initial [Ca(2+)](i) oscillation. However, only 54% of the cells that responded initially displayed subsequent [Ca(2+)](i) oscillations during long-term flow and the magnitude of the subsequent oscillations was only 28% of the initial response.     

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE(2) but no change in mineralization.

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE(2) production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE(2) is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 +/- 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE(2) production. After 7 days of static culture (0 dyn/cm(2)) or low (0.06 +/- 0.05 Pa, 0.3 Hz) or high (0.6 +/- 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference (P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE(2) levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.     

Cycle number and waveform of fluid flow affect bovine articular chondrocytes

Many studies have shown that a loading-induced (bio)physical signal regulates chondrocyte behavior. In a recent study our group has demonstrated the shear stress level- and frequency-dependent effect of sinusoidal oscillatory fluid flow on bovine articular chondrocyte (BAC) cytosolic calcium concentration ([Ca(2+)](i)), neglecting the fact that chondrocytes are not likely to see these ideal waveform in vivo or in vitro. Furthermore, possible overload of articular cartilage or excessive shear stress in chondrocyte cultures are more likely to be of a short nature. Therefore, in this study we choose to investigate a saw-tooth waveform oscillating fluid flow at varying exposure times in comparison to the established sinusoidal oscillatory waveform. [Ca(2+)](i), as an early signaling molecule, was quantified using the fluorescent dye fura-2. BAC were exposed to 1 Hz sinusoidal or saw-tooth waveform oscillating fluid flow at 2.2 Pa flow rates in a parallel plate flow chamber for 8 different loading times. As little as 5 cycles of oscillatory fluid flow were sufficient to increase [Ca(2+)](i) significantly over baseline. The number of responding cells could not be increased any further after a sufficient number of cycles (11), regardless of the waveform. Furthermore, a saw-tooth waveform appeared to be more stimulatory than regular sinusoidal oscillating flow at higher cycle numbers. BAC appear to be able to respond to these biophysical stimuli in a differentiated manner. This ability might give every single chondrocyte the capability to maintain its territory autonomously, since chondrocytes distributed in articular cartilage without the possibility to interact, e.g., via cell processes.     

P2Y purinoceptors are responsible for oscillatory fluid flow-induced intracellular calcium mobilization in osteoblastic cells

We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca(2+)(i) mobilization via the inositol 1,4,5-trisphosphate pathway in the presence of 2% fetal bovine serum (FBS). However, the molecular mechanism of fluid flow-induced Ca(2+)(i) mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca(2+)](i) in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5' nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca(2+)](i) in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca(2+)](i), suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca(2+)](i). Furthermore, adenosine, ADP, UDP, or adenosine 5'-O-(3-thiotriphosphate) did not induce Ca(2+)(i) mobilization under oscillatory fluid flow without FBS. Pyridoxal phosphate 6-azophenyl-2,4'-disulfonic acid, an antagonist of P2X purinoceptors, did not alter the effect of fluid flow on the Ca(2+)(i) response, whereas pertussis toxin, a G(i/o)-protein inhibitor, inhibited fluid flow-induced increases in [Ca(2+)](i) in the presence of 2% FBS. Thus, by the process of elimination, our data suggest that P2Y purinoceptors (P2Y2 or P2Y4) are involved in the Ca(2+)(i) response to fluid flow. Finally, a decreased percentage of MC3T3-E1 osteoblastic cells treated with P2Y2 antisense oligodeoxynucleotides responded to fluid flow with an increase in [Ca(2+)](i), and an increased percentage of ROS 17/2.8 cells, which do not normally express P2Y2 purinoceptors, transfected with P2Y2 purinoceptors responded to fluid flow in the presence of 2% FBS, confirming that P2Y2 purinoceptors are responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization in osteoblastic cells.     

Fluid flow induced calcium response in osteoblasts: mathematical modeling

Fluid flow in the bone lacuno-canalicular network can induce dynamic fluctuation of intracellular calcium concentration ([Ca(2+)](i)) in osteoblasts, which plays an important role in bone remodeling. There has been limited progress in the mathematical modeling of this process probably due to its complexity, which is controlled by various factors such as Ca(2+) channels and extracellular messengers. In this study we developed a mathematical model to describe [Ca(2+)](i) response induced by fluid shear stress (SS) by integrating the major factors involved and analyzed the effects of different experimental setups (e.g. [Ca(2+)](i) baseline, pretreatment with ATP). In this model we considered the ATP release process and the activities of multiple ion channels and purinergic receptors. The model was further verified quantitatively by comparing the simulation results with experimental data reported in literature. The results showed that: (i) extracellular ATP concentration has more significant effect on [Ca(2+)](i) baseline (73% increase in [Ca(2+)](i) with extracellular ATP concentration varying between 0 and 10 μM), as compared to that induced by SS (25% variation in [Ca(2+)](i) with SS varying from 0 to 3.5 Pa); (ii) Pretreatment with ATP-medium results in different [Ca(2+)](i) response as compared to the control group (ATP-free medium) under SS; (iii) Relative [Ca(2+)](i) fluctuation over baseline is more reliable to show the [Ca(2+)](i) response process than the absolute [Ca(2+)](i) response peak. The developed model may improve the experimental design and facilitate our understanding of the mechanotransduction process in osteoblasts.     

Invited review: effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo.

To study the effects of flow on in situ endothelial intracellular calcium concentration ([Ca(2+)](i)) signaling, rat aortic rings were loaded with fura 2, mounted on a tissue flow chamber, and divided into control and flow-pretreated groups. The latter was perfused with buffer at a shear stress of 50 dyns/cm(2) for 1 h. Endothelial [Ca(2+)](i) responses to ACh or shear stresses were determined by ratio image analysis. Moreover, ACh-induced [Ca(2+)](i) elevation responses were measured in a calcium-free buffer, or in the presence of SKF-96365, to elucidate the role of calcium influx in the flow effects. Our results showed that 1) ACh increased endothelial [Ca(2+)](i) in a dose-dependent manner, and these responses were incremented by flow-pretreatment; 2) the differences in ACh-induced [Ca(2+)](i) elevation between control and flow-pretreated groups were abolished by SKF-96365 or by Ca(2+)-free buffer; and 3) in the presence of 10(-5) M ATP, shear stress induced dose-dependent [Ca(2+)](i) elevation responses that were not altered by flow-pretreatment. In conclusion, flow-pretreatment augments the ACh-induced endothelial calcium influx in rat aortas ex vivo.     

Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Inhibition of ADP-induced intracellular Ca2+ responses and platelet aggregation by the P2Y12 receptor antagonists AR-C69931MX and clopidogrel is enhanced by prostaglandin E1

P2Y(12) antagonists such as clopidogrel and AR-C69931MX inhibit aggregation by antagonizing the effects of ADP at P2Y(12) receptors on platelets. Agents such as PGE(1) also inhibit aggregation by stimulating adenylate cyclase to produce cAMP, which interferes with Ca(2+) mobilization within the cell. Since one facet of P2Y(12) receptors is that they mediate inhibition of adenylate cyclase by ADP, it might be expected that P2Y(12) antagonists would interact with PGE(1). We have explored the effects of PGE(1) and AR-C69931MX singly and in combination on ADP-induced intracellular Ca(2+) ([Ca(2+)](i)) responses and aggregation. PGE(1) alone caused parallel dose-dependent inhibition of [Ca(2+)](i) and aggregation responses. AR-C66931MX alone caused only partial inhibition of [Ca(2+)](i) despite a marked inhibitory effect on aggregation. Combinations of PGE(1) with AR-C66931MX were found to act in synergy to reduce both [Ca(2+)](i) and aggregation. This effect was confirmed in patients with acute coronary syndromes by studying the inhibitory effects of PGE(1) on [Ca(2+)](i) and aggregation before and after clopidogrel. In summary, we have shown that P2Y(12) antagonists interact with natural agents such as PGE(1) to provide more effective inhibition of [Ca(2+)](i) and platelet aggregation. This would contribute to the effectiveness of P2Y(12) antagonists as antithrombotic agents in man.     

Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE(2), which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in hepatic parenchymal cells by agonists released during stress, such as with PGE(2) and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca(2+)](i) in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE(2) caused a dose-dependent increase in [Ca(2+)](i) from basal values of 130 +/- 10 to maximal levels of 434 +/- 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca(2+) dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca(2+) from intracellular stores, also partially blocked the increase in [Ca(2+)](i) caused by PGE(2), suggesting that intracellular Ca(2+) pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca(2+) pools are involved in the increase in [Ca(2+)](i) caused by PGE(2). Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca(2+)](i) due to PGE(2) in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl(-) was omitted, glycine was much less effective in preventing the increase in [Ca(2+)](i) due to PGE(2). Phenylephrine, an alpha(1)-type adrenergic receptor agonist, also increased [Ca(2+)](i), as expected, from 159 +/- 20 to 432 +/- 43 nM. Glycine also blocked the increase in [Ca(2+)](i) due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca(2+)](i) in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.     

Polycystin 2 is involved in the nitric oxide production in responding to oscillating fluid shear in MLO-Y4 cells

As a mechano-calcium channel, polycystin2 (PC2) play an important role in the response of renal epithelial cells to fluid flow shear stress. In bone tissue, osteocytes are well known as the main mechanosensory cells, and sensitive to fluid flow stimulus in vitro. In the study, we investigated the effects of oscillating fluid flow (OFF, 2 h, 1 Hz, 1.0 Pa) on the release of Nitric Oxide (NO) and ProstaglandinE2 (PGE2), and the role of PC2 on the release. Our findings demonstrate that PC2 expression increases after 2 h of OFF, and silencing PC2 by RNAi inhibits downstream NO production and iNOS expression, but does not affect the response of PGE2 to OFF.     

Modulation of Ca2+ transients in cultured endothelial cells in response to fluid flow through alphav integrin

In order to determine whether integrin dynamics is associated with intracellular Ca(2+) concentration ([Ca(2+)](i)) mobilization in ECs in response to hemodynamic forces, changes in [Ca(2+)](i) in fluo-4-loaded cultured bovine aortic endothelial cells (BAECs) under fluid flow conditions were visualized employing laser scanning confocal microscopy. Following the onset of flow stimulus, transient increases in [Ca(2+)](i) occurred several times in individual BAECs during the 30-min observation period. The frequency of these [Ca(2+)](i) transients was clearly reduced by the application of an integrin antagonist (GRGDSP peptide). Furthermore, treatment of cells with an integrin activator (Mn(2+)) resulted in reduction of peak [Ca(2+)](i) levels and elevated frequency, which was markedly rescued upon GRGDSP administration. In contrast, an actin de-polymerizing agent (cytochalasin D) exerted no inhibitory effects; rather, cytochalasin D more likely facilitated [Ca(2+)](i) transients. Moreover, [Ca(2+)](i) transients, which were suppressed by short interference RNA-induced silencing of alphav integrin, exhibited greater frequently in cells cultured on vitronectin substratum in comparison with those cultured on fibronectin or collagen substratum. Either removal of extracellular Ca(2+), application of an inhibitor of endoplasmic reticulum Ca(2+)-ATPase (thapsigargin) or non-selective cation channel blocker (La(3+)) inhibited the [Ca(2+)](i) transients. Additionally, [Ca(2+)](i) transients were attenuated by extracellular signal-regulated kinase (ERK) kinase inhibitor (U0126); in contrast, [Ca(2+)](i) transients were unaffected by tyrosine kinase inhibitor (genistein) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Therefore, our findings revealed that alphav integrin dynamics modulates the frequency of flow-induced [Ca(2+)](i) transients in BAECs in an ERK-dependent fashion.     

Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow

Although it is well accepted that bone tissue metabolism is regulated by external mechanical loads, it remains unclear to what load-induced physical signals bone cells respond. In this study, a novel computer-controlled stretch device and parallel plate flow chamber were employed to investigate cytosolic calcium (Ca2+i) mobilization in response to a range of dynamic substrate strain levels (0.1-10 percent, 1 Hz) and oscillating fluid flow (2 N/m2, 1 Hz). In addition, we quantified the effect of dynamic substrate strain and oscillating fluid flow on the expression of mRNA for the bone matrix protein osteopontin (OPN). Our data demonstrate that continuum strain levels observed for routine physical activities (< 0.5 percent) do not induce Ca2+i responses in osteoblastic cells in vitro. However, there was a significant increase in the number of responding cells at larger strain levels. Moreover, we found no change in osteopontin mRNA level in response to 0.5 percent strain at 1 Hz. In contrast, oscillating fluid flow predicted to occur in the lacunar-canalicular system due to routine physical activities (2 N/m2, 1 Hz) caused significant increases in both Ca2+i and OPN mRNA. These data suggest that, relative to fluid flow, substrate deformation may play less of a role in bone cell mechanotransduction associated with bone adaptation to routine loads.     

Integrin mobilizes intracellular Ca(2+) in renal vascular smooth muscle cells

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca(2+) signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca(2+)](i) rapidly increased from 91 +/- 4 to 287 +/- 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca(2+) mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca(2+)](i), which propagated as Ca(2+) waves traveling across the cell and induced a rapid elevation of nuclear [Ca(2+)](i). Spontaneous recurrence of smaller-amplitude Ca(2+) waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca(2+) channels with nifedipine or removal of extracellular Ca(2+) did not inhibit the RGD-induced Ca(2+) mobilization. However, pretreatment of 20 microM ryanodine completely eliminated the RGD-induced Ca(2+) mobilization. Anti-beta(1) and anti-beta(3)-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca(2+)](i). Incubation with anti-beta(1)- or beta(3)-integrin antibodies inhibited the increase in [Ca(2+)](i) induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca(2+) from ryanodine-sensitive Ca(2+) stores in renal VSMC via integrins, which can trigger cytoplasmic Ca(2+) waves propagating throughout the cell.     

Flow-induced prostaglandin E2 release regulates Na and K transport in the collecting duct

Fluid shear stress (FSS) is a critical regulator of cation transport in the collecting duct (CD). High-dietary sodium (Na) consumption increases urine flow, Na excretion, and prostaglandin E(2) (PGE(2)) excretion. We hypothesize that increases in FSS elicited by increasing tubular flow rate induce the release of PGE(2) from renal epithelial cells into the extracellular compartment and regulate ion transport. Media retrieved from CD cells exposed to physiologic levels of FSS reveal several fold higher concentration of PGE(2) compared with static controls. Treatment of CD cells with either cyclooxygenase-1 (COX-1) or COX-2 inhibitors during exposure to FSS limited the increase in PGE(2) concentration to an equal extent, suggesting COX-1 and COX-2 contribute equally to FSS-induced PGE(2) release. Cytosolic phospholipase A2 (cPLA2), the principal enzyme that generates the COX substrate arachidonic acid, is regulated by mitogen-activated protein-kinase-dependent phosphorylation and intracellular Ca(2+) concentration ([Ca(2+)](i)), both signaling processes, of which, are activated by FSS. Inhibition of the ERK and p38 pathways reduced PGE(2) release by 53.3 ± 8.4 and 32.6 ± 11.3%, respectively, while antagonizing the JNK pathway had no effect. In addition, chelation of [Ca(2+)](i) limited the FSS-mediated increase in PGE(2) concentration by 47.5 ± 7.5% of that observed in untreated sheared cells. Sheared cells expressed greater phospho-cPLA2 protein abundance than static cells; however, COX-2 protein expression was unaffected (P = 0.064) by FSS. In microperfused CDs, COX inhibition enhanced flow-stimulated Na reabsorption and abolished flow-stimulated potassium (K) secretion, but did not affect ion transport at a slow flow rate, implicating that high tubular flow activates autocrine/paracrine PGE(2) release and, in turn, regulates flow-stimulated cation transport. In conclusion, FSS activates cPLA2 to generate PGE(2) that regulates flow-mediated Na and K transport in the native CD. We speculate that dietary sodium intake modulates tubular flow rate to regulate paracrine PGE(2) release and cation transport in the CD.     

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.     

Membrane potential dependent modulations of calcium oscillations in insulin-secreting INS-1 cells

This study was undertaken to examine the role of K(+) channels on cytosolic Ca(2+) ([Ca(2+)](i)) in insulin secreting cells. [Ca(2+)](i) was measured in single glucose-responsive INS-1 cells using the fluorescent Ca(2+) indicator Fura-2. Glucose, tolbutamide and forskolin elevated [Ca(2+)](i) and induced [Ca(2+)] oscillations. Whereas the glucose effect was delayed and observed in 60% and 93% of the cells, in a poorly and a highly glucose-responsive INS-1 cell clone, respectively, tolbutamide and forskolin increased [Ca(2+)](i) in all cells tested. In the latter clone, glucose induced [Ca(2+)](i) oscillations in 77% of the cells. In 16% of the cells a sustained rise of [Ca(2+)](i) was observed. The increase in [Ca(2+)](i) was reversed by verapamil, an L-type Ca(2+) channel inhibitor. Adrenaline decreased [Ca(2+)](i) in oscillating cells in the presence of low glucose and in cells stimulated by glucose alone or in combination with tolbutamide and forskolin. Adrenaline did not lower [Ca(2+)](i) in the presence of 30mM extracellular K(+), indicating that adrenaline does not exert a direct effect on Ca(2+) channels but increases K(+) channel activity. As for primary b-cells, [Ca(2+)](i) oscillations persisted in the presence of closed K(ATP) channels; these also persisted in the presence of thapsigargin, which blocks Ca(2+) uptake into Ca(2+) stores. In contrast, in voltage-clamped cells and in the presence of diazoxide (50mM), which hyperpolarizes the cells by opening K(ATP) channels, [Ca(2+)](i) oscillations were abolished. These results support the hypothesis that [Ca(2+)](i) oscillations depend on functional voltage-dependent Ca(2+) and K(+) channels and are interrupted by a hyperpolarization in insulin-secreting cells.     

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

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

Activating of ATP-dependent K+ channels comprised of K(ir) 6.2 and SUR 2B by PGE2 through EP2 receptor in cultured interstitial cells of Cajal from murine small intestine.

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE(2) might affect the electrical properties of cultured ICC by activating ATP-dependent K(+) channels and, the EP receptor subtypes and the subunits of ATP-dependent K(+) channels involved in these activities were identified. In addition, the regulation of intracellular Ca(2+) ([Ca(2+)](i)) mobilization may be involved the action of PGE(2) on ICC. Treatments of ICC with PGE(2) inhibited electrical pacemaker activities in the same manner as pinacidil, an ATP-dependent K(+) channel opener and PGE(2) had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K(+) channels exist in ICC and that these are composed of K(ir) 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP(2) receptor agonist) showed the actions on pacemaker currents in the same manner as PGE(2). However sulprostone (a mixed EP(1) and EP(3) agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP(2) receptor in ICC. To investigate cAMP involvement in the effects of PGE(2) on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE(2) on pacemaker currents, and PGE(2) did not stimulate cAMP production. Also, we found PGE(2) inhibited the spontaneous [Ca(2+)](i) oscillations in cultured ICC. These observations indicate that PGE(2) alters pacemaker currents by activating the ATP-dependent K(+) channels comprised of K(ir) 6.2-SUR 2B in ICC and this action of PGE(2) are through EP(2) receptor subtype and also the activation of ATP-dependent K(+) channels involves intracellular Ca(2+) mobilization.