Fura-2 Fluorescence Research Articles

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.

Diminished cardiac contractile response to tetrahydropapaveroline in hypertension: role of β-adrenoceptors and intracellular Ca 2+

Tetrahydropapaveroline (THP), a condensation product of ethanol-derived acetaldehyde, potentiates cardiac function through a β-adrenergic mechanism. It is well established that β-adrenergic activity is markedly depressed in hypertension. However, little is known about the myocardial action of THP in hypertension. In this study, the effect of THP was examined using left ventricular papillary muscles and ventricular myocytes from 10-week-old normotensive (WKY) and spontaneously hypertensive (SHR) rats. The mechanical parameters evaluated include: peak tension developed (PTD), peak twitch amplitude (PTA), time-to-PTD/PTA (TPT/TPS), time-to-90% relaxation/relengthening (RT 90/TR 90), and the maximal velocities of contraction/shortening and relaxation/relengthening (±VT/±d L/d t). Intracellular Ca 2+ transients were measured as fura-2 fluorescence intensity changes (ΔFFI). THP (0.01–100 μM) produced a concentration-dependent increase in myocardial contraction on muscles and myocytes from both groups of animals. However, preparations from the SHR group were generally less responsive to THP than their normotensive counterparts. The increase in contractility by THP was associated with increases in ΔFFI and ±VT, and shortening of TPT/TPS and RT 90/TR 90. The role of β-adrenoceptor(s) in the mechanism of action of THP was explored using specific β-receptor subtype antagonists CGP 207.12A (β 1) and ICI 118,551 (β 2). In preparations from both WKY and SHR hearts, the THP-induced increase in cardiac contractility was either attenuated or blocked by CGP 207.12A and ICI 118,551. These results indicate that THP exhibits a positive action on myocardial contraction that is mediated, in part, through both β 1 and β 2 adrenergic receptors. This cardiac inotropic response, however, is markedly diminished in hypertension, which is due possibly to alterations in β-adrenergic signal transduction.

Ca2 mobilization induced by δ‐hexachlorocyclohexane in Madin Darby canine kidney cells

The effect of the pesticide δ-hexachlorocyclohexane (δ-HCH) were examined on Ca2+ signaling in Madin Darby canine kidney (MDCK) using fura-2 as a Ca2+ probe. δ-HCH at concentrations of 5–200 mM increased intracellular free Ca2+ concentration ([Ca2+]i) concentration-dependently. The [Ca2+]i increase comprised an immediate rise followed by a sustained phase within 5 min of measurement. External Ca2+ removal slightly reduced the [Ca2+]i increase. In Ca2+-free medium, 150 μM δ-HCH did not increase [Ca2+]i after pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and two endoplasmic reticulum (ER) Ca2+ pump inhibitors, thapsigargin (1 μM) and cyclopiazonic acid (100 μM). Conversely, pretreatment with δ-HCH prevented thapsigargin, cyclopiazonic acid, and CCCP from releasing more Ca2+, suggesting 150 μM δ-HCH released Ca2+ from the ER and mitochondria. δ-HCH (150 μM) activated Mn2+ quench of fura-2 fluorescence, confirming that δ-HCH induced Ca2+ influx. Addition of 3 mM Ca2+ induced a concentration-dependent [Ca2+]i increase after pretreatment with 100–200 μM δ-HCH for 870 sec in Ca2+-free medium. The δ-HCH (150 μM)-induced Ca2+ release was decreased by inhibiting phospholipase C with 1 μM U73122. Collectively, we have found that δ-HCH increased [Ca2+]i in MDCK cells by releasing Ca2+ from the ER and mitochondria, followed by capacitative Ca2+ entry. Drug Dev. Res. 50:186–192, 2000. © 2000 Wiley-Liss, Inc.

The involvement of intracellular Ca(2+) in 5-HT(1B/1D) receptor-mediated contraction of the rabbit isolated renal artery.

5-Hydroxytryptamine(1B/1D) (5-HT(1B/1D)) receptor coupling to contraction was investigated in endothelium-denuded rabbit isolated renal arteries, by simultaneously measuring tension and intracellular [Ca(2+)], and tension in permeabilized smooth muscle cells. In intact arterial segments, 1 nM - 10 microM 5-HT failed to induce contraction or increase the fura-2 fluorescence ratio (in the presence of 1 microM ketanserin and prazosin to block 5-HT(2) and alpha(1)-adrenergic receptors, respectively). However, in vessels pre-exposed to either 20 mM K(+) or 30 nM U46619, 5-HT stimulated concentration-dependent increases in both tension and intracellular [Ca(2+)]. 1 nM - 10 microM U46619 induced concentration-dependent contractions. In the presence of nifedipine (0.3 and 1 microM) the maximal contraction to U46619 (10 microM) was reduced by around 70%. The residual contraction was abolished by the putative receptor operated channel inhibitor, SKF 96365 (2 microM). With 0.3 microM nifedipine present, 100 nM U46619 evoked similar contraction to 30 nM U46619 in the absence of nifedipine, but contraction to 5-HT (1 nM - 10 microM) was abolished. In permeabilized arterial segments, 10 mM caffeine, 1 microM IP(3) or 100 microM phenylephrine, each evoked transient contractions by releasing Ca(2+) from intracellular stores, whereas 5-HT had no effect. In intact arterial segments pre-stimulated with 20 mM K(+), 5-HT-evoked contractions were unaffected by 1 microM thapsigargin, which inhibits sarco- and endoplasmic reticulum calcium-ATPases. In vessels permeabilized with alpha-toxin and then pre-contracted with Ca(2+) and GTP, 5-HT evoked further contraction, reflecting increased myofilament Ca(2+)-sensitivity. Contraction linked to 5-HT(1B/1D) receptor stimulation in the rabbit renal artery can be explained by an influx of external Ca(2+) through voltage-dependent Ca(2+) channels and sensitization of the contractile myofilaments to existing levels of Ca(2+), with no release of Ca(2+) from intracellular stores.

Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate.

Intercellular propagation of signals through connexin32-containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin-transfected HeLa cells was standardized by measuring the spreading of microinjected Mn(2+) that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca(2+) wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32-containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.

Effect of urea and osmotic cell shrinkage on Ca2+ entry and contraction of vascular smooth muscle cells

The present study was performed to elucidate the effects of urea on vascular smooth muscle cells (SMC). Addition of urea (20, 50, 100 mM) to physiological salt solution blunted the vasoconstrictory effect of phenylephrine (by 17, 25 and 30%, respectively) and of an increased extracellular K+ concentration (by 7, 14 and 19%, respectively) without affecting the basal tone of rabbit arterial rings. According to Fura-2 fluorescence in cultured SMC (A7r5), urea had no effect on basal intracellular calcium activity ([Ca2+]i), but significantly blunted the increase of [Ca2+]i following an increase of extracellular K+. Whole-cell patch-clamp studies revealed that the Ca2+ current through voltage-sensitive Ca2+ channels is significantly inhibited in the presence of urea. As evident from calcein fluorescence, addition of urea leads to sustained cell shrinkage. The effects of urea on vascular tone, [Ca2+]i activity, voltage-gated Ca2+ channels and cell volume are mimicked by addition of raffinose or NaCl. However, the cell shrinkage induced by urea is sustained, whereas the addition of equiosmolar NaCl is only transient and followed by a regulatory cell volume increase. Moreover, hypertonic NaCl increases, whereas urea decreases, the transcription of cell-volume-regulated kinase hsgk. In conclusion, urea leads to sustained shrinkage of vascular smooth muscle cells, which is followed by inhibition of voltage-gated Ca2+ channels, a decrease of [Ca2+]i and thus blunts the vasoconstrictory action of phenylephrine and increased extracellular K+ concentration.

Selective measurement of endothelial or smooth muscle [Ca 2+] i in pressurized/perfused cerebral arteries with fura-2

Despite a critical role for calcium in endothelial regulation of cerebrovascular tone, endothelial intracellular calcium ([Ca 2+] i) has never been measured in the context of an intact pressurized cerebral vessel. The purpose of the present study was to selectively measure endothelial or smooth muscle [Ca 2+] i and diameter in a pressurized/perfused cerebral vessel. In a pressurized rat middle cerebral artery, fura-2 AM was administered selectively to either the luminal (endothelium) or abluminal (smooth muscle) side of the vessel. Selectivity of loading was determined by measuring fura-2 fluorescence before and after removal of the endothelium. Removal of the endothelium virtually eliminated fura-2 fluorescence. In addition, 2-methylthioadenosine triphosphate (2MeS-ATP, a selective endothelial P2 receptor agonist) was used to infer the selectivity of fura-2 loading. It was reasoned that 2MeS-ATP should produce a decrease in smooth muscle [Ca 2+] i and an increase in endothelial [Ca 2+] i in selectively loaded vessels, consistent with its role as an NO-dependent dilator. In smooth muscle loaded vessels, [Ca 2+] i went from 252±8 to 82±9 nM following luminal administration of 2MeS-ATP, whereas in endothelial loaded vessels, [Ca 2+] i went from 137±11 to 271±20 nM. Thus, a method is provided which allows for selective measurement of endothelial or smooth muscle [Ca 2+] i with simultaneous measurement of diameter in a pressurized cerebral vessel.

Attenuated cardiac contractile responsiveness to insulin-like growth factor I in ventricular myocytes from biobreeding spontaneous diabetic rats.

Insulin-like growth factor I (IGF-1) stimulates cardiac growth and contraction, but resistance to its action has been reported in diabetes. This study was to determine if IGF-1-induced cardiac contractile action is altered in rats genetically predisposed to diabetes. Ventricular myocytes were isolated from spontaneously biobreeding diabetes-prone (BB/DP) rats and their diabetes-resistant littermates (BB/DR). Mechanical properties were evaluated in cardiomyocytes using a video-based edge-detection system. Myocytes were electrically stimulated at 0.5 Hz. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR(90)). Intracellular Ca(2+) transients were measured as changes in fura-2 fluorescence intensity (DeltaFFI). Myocytes from BB/DP rats displayed increased PS, prolonged TPS and TR(90,) as well as reduced resting FFI compared to the BB/DR group. IGF-1 (10(-10)-10(-6) M) caused a dose-dependent increase in PS in myocytes from BB/DR but not BB/DP rats. The increase of PS was blunted by IGF-1 antagonist H-1356, phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, but not tyrosine kinase inhibitor genistein. None of these agents affected responses to IGF-1 in BB/DP myocytes. Interestingly, IGF-1 elicited a comparable dose-dependent increase in Ca(2+) transients in myocytes from both BB/DR and BB/DP rats. These results suggest that the attenuation of IGF-1-induced cardiac contractile response in chemically-induced diabetes also exists in diabetes of genetic origin, possibly due to mechanisms involving PI-3 kinase and intracellular Ca(2+) sensitivity.

Novel effects of clotrimazole on Ca 2+ signaling in Madin Darby canine kidney cells

The effect of clotrimazole on Ca 2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca 2+ indicator. Clotrimazole (1–30 μM) induced a concentration-dependent [Ca 2+] i increase. The [Ca 2+] i increase comprised an initial rise and a slow decay. External Ca 2+ removal partly inhibited the Ca 2+ signals by reducing both the initial rise and the decay phase, indicating that clotrimazole triggered both Ca 2+ influx and Ca 2+ release. Pretreatment with 30 μM clotrimazole in Ca 2+-free medium abolished the Ca 2+ release induced by thapsigargin (1 μM), an endoplasmic reticulum Ca 2+ pump inhibitor, and conversely, pretreatment with thapsigargin prevented clotrimazole from releasing more Ca 2+. This suggests that the thapsigargin-sensitive Ca 2+ store is the source of clotrimazole-induced Ca 2+ release. Clotrimazole (10 μM) triggered Mn 2+ quench of fura-2 fluorescence which was partly inhibited by 1 mM La 3+. Addition of 3 mM Ca 2+ induced a [Ca 2+] i increase after preincubation with 10 μM clotrimazole in Ca 2+-free medium, indicating that clotrimazole activated capacitative Ca 2+ entry. However, 10 and 30 μM clotrimazole inhibited 1 μM thapsigargin-induced capacitative Ca 2+ entry by 21% and 74%, respectively. Pretreatment with 40 μM aristolochic acid to inhibit phospholipase A 2 reduced 30 μM clotrimazole-induced Ca 2+ release by 51%, but inhibiting phospholipase C with 2 μM U73122 had little effect. This implies that clotrimazole induces Ca 2+ release in an IP 3-independent manner, which could be modulated by phospholipase A 2-coupled events.

A Fundamental Role for the Nitric Oxide-G-Kinase Signaling Pathway in Mediating Intercellular Ca2+Waves in Glia

In this study, we highlight a role for the nitric oxide-cGMP-dependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca(2+) wave initiation and propagation. Addition of the NO donor molsidomine (100-500 microM) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca(2+)](i) in individual cells and also local intercellular Ca(2+) waves, which persisted after removal of extracellular Ca(2+). High concentrations of ryanodine (100-200 microM) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca(2+)](i), indicating that NO mobilizes Ca(2+) from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 microM nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca(2+)](i), and inhibition of Mn(2+) quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 microM aqueous NO to cells, indicating that NO also induces Ca(2+) influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca(2+) waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced Delta[Ca(2+)](i) and the rate of Ca(2+) wave propagation in these cultures. Results from this study suggest that NO-G-kinase signaling is coupled to Ca(2+) mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca(2+) waves in glia.

P2 Purinoceptor expression and functional changes of hypoxia-activated cultured rat retinal microglia.

P(2) purinoceptors appear to modulate microglia function, but their role in hypoxic microglia has not been investigated. We examined in postnatal rat retinal microglia cultured under hypoxic (1% oxygen) condition, their P2 expression, proliferation and cytokine release in the presence or absence of the P2 receptor agonists and antagonists. Fura-2 fluorescence measurements of intracellular Ca(2+) rises to P2 receptor agonists and antagonists indicated that both P(2U) and P(2Z) were expressed in hypoxic microglia. Hypoxia induced BrdU incorporation and release of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as well. The P(2U) agonist, UTP, maintained the BrdU incorporation, whereas the P(2Z) agonist, BzATP, suppressed it, but significantly enhanced IL-1beta and TNF-alpha release, suggesting that the P(2U) response may underlie the mitotic activity, and that of P(2Z), the IL-1beta and TNF-alpha release of hypoxia-activated microglia.

Diabetes Enhances Acetaldehyde-Induced Depression of Cardiac Myocyte Contraction

It is well established that cardiomyopathy is a consistent feature of diabetes and that alcohol consumption increases the risk of cardiovascular disease among diabetic subjects. Acetaldehyde (ACA), the main ethanol metabolite, is considered to play a role in the ethanol-induced cardiac dysfunction. It has been reported recently that the negative inotropic effect of ACA was more potent in the diabetic myocardium. To determine whether the disparate ACA-induced myocardial depression in diabetes is due to intrinsic alterations at the cellular level, mechanical properties in response to ACA were evaluated in ventricular myocytes from both normal and streptozotocin-induced diabetic rat hearts. Myocytes were electrically stimulated to contract at 0.5 Hz and contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocities of shortening and relengthening (±dL/dt). Ca2+ transients were measured as fura-2 fluorescence intensity (ΔFFI) changes. ACA (0.1–30 mM) disproportionately depressed PS in a dose-dependent manner, in myocytes from diabetic hearts compared to normal hearts. Interestingly, the degree of inhibition in ΔFFI was similar in both groups. Neither the duration nor maximal velocities of shortening and relengthening were affected by ACA in either group. These results are the first to suggest that enhanced ACA-induced myocardial depression in diabetes is due to disparate intrinsic actions on individual myocytes. The mechanism underlying the alteration of ACA-induced myocardial depression may be due, in part, to depressed Ca2+ responsiveness in diabetic hearts.

Mechanisms of nordihydroguaiaretic acid-induced [Ca 2+], increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca 2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 μM) increased [Ca 2+] i concentration-dependently. The [Ca 2+] i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca 2+ removal partly inhibited the Ca 2+ signals induced by 25–100 μM NDGA and abolished that induced by 10 μM NDGA. In Ca 2+-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca 2+] i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorpphenylhydrazone (CCCP; 2 μM) and the endoplasmic reticulum (ER) Ca 2+ pump inhibitor thapsigargin (1 μM). However, 0.1 mM NDGA still increased [Ca 2+] i after Ca 2+ stores had been depleted by pretreating with 2 μM CCCP, 1 μM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 μM) activated Mn 2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 μM La 3+. This implies NDGA induced Ca 2+ influx mainly via a La 3+-sensitive pathway. Consistently, 50 μM La 3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca 2+] i increase. Adding 3 mM Ca 2+ increased [Ca 2+] i in cells pretreated with 0.1 mM NDGA in Ca 2+-free medium, suggesting NDGA activated capacitative Ca 2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca 2+ did not alter 1 μM thapsigargin-induced capacitative Ca 2+ entry. Pretreatment with 40 μM aristolochic acid to inhibit phospholipase A 2 reduced 0.1 mM NDGA-induced Ca 2+ release by 65%, but inhibiting phospholipase C with 2 μM U73122 had little effect. This suggests NDGA-induced Ca 2+ release was independent of inositol 1,4,5-trisphosphate (IP 3), but was modulated by phospholipase A 2.

A novel action of the antianginal drug bepredil: induction of internal Ca 2+ release and external Ca 2+ influx in Madin–Darby canine kidney (MDCK) epithelial cells

The effect of the antianginal drug bepridil on Ca 2+ signaling in Madin–Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca 2+ probe. Bepridil at 10–50 μM evoked a significant rise in cytosolic free Ca 2+ concentration ([Ca 2+] i) in a dose-dependent manner. The [Ca 2+] i rise consisted of an immediate initial rise and a slow decay. Removal of external Ca 2+ partly inhibited the Ca 2+ signals by reducing both the initial rise and the decay phase, suggesting that bepridil activated both external Ca 2+ influx and internal Ca 2+ release. In Ca 2+-free medium, pretreatment with 50 μM bepridil nearly abolished the Ca 2+ release induced by thapsigargin (1 μM), an endoplasmic reticulum Ca 2+ pump inhibitor, and vice versa, pretreatment with thapsigargin inhibited most of the bepridil-induced Ca 2+ release, suggesting that the thapsigargin-sensitive Ca 2+ store was the main source of bepridil-induced Ca 2+ release. Bepridil (50 μM) induced considerable Mn 2+ quench of fura-2 fluorescence at an excitation wavelength of 360 nm, which was partly inhibited by La 3+ (0.1 mM). Consistently, La 3+ (0.1 mM) pretreatment significantly inhibited the bepridil-induced [Ca 2+] i rise. Addition of 3 mM Ca 2+ induced a significant [Ca 2+] i rise after prior incubation with 10–50 μM bepridil in Ca 2+-free medium, suggesting that bepridil induced dose-dependent capacitative Ca 2+ entry. However, 50 μM bepridil inhibited 1 μM thapsigargin-induced capacitative Ca 2+ entry by 38%. Pretreatment with aristolochic acid (40 μM) so as to inhibit phospholipase A 2 inhibited 50 μM bepridil-induced internal Ca 2+ release by 42%, but inhibition of phospholipase C with U73122 (2 μM) or inhibition of phospholipase D with propranolol (0.1 mM) had little effect, suggesting that bepridil induced internal Ca 2+ release in an inositol 1,4,5-trisphosphate-independent manner that could be modulated by phospholipase A 2-coupled events. This is the first report providing evidence that bepridil, currently used as an antianginal drug, induced a rise in [Ca 2+] i in a non-excitable cell line.

Trp1, a Candidate Protein for the Store-operated Ca2+Influx Mechanism in Salivary Gland Cells

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Acute triiodothyronine administration does not reverse depressed contractile performance following catecholamine exposure in isolated rat cardiomyocytes.

It has been previously suggested that triiodothyronine (T3) may reverse depressed cardiac contractile performance occurring after excessive catecholamine stimulation. We therefore investigated the effects of T3 on intracellular calcium transients and contractile performance in isolated ventricular rat myocytes. Isolated rat myocytes were loaded with the calcium indicator FURA-2/AM (50 micromol/L) and superfused with Krebs-Henseleit solution (pH 7.4). Cells were illuminated by ultraviolet light and fluorescent images obtained with a target camera at 340 nm and 380 nm excitation wavelengths (ratio method). Simultaneous measurements of calcium transients and cell shortening (35 degrees C, electrical field stimulation: 0.5 Hz) were done. At steady state conditions, FURA-2 loaded myocytes were superfused for 60 min with epinephrine (0.1 micromol/L). After 60 min the effect of T3 (10 micromol/L) on calcium transients and shortening were measured. As control, only the effect of 10 micromol/L T3 was analyzed. Epinephrine significantly increased cell shortening and FURA-2 signals to 148.6+/-8.8% and 109.5+/-3.1% (p < 0.01: n = 21; 5 min) respectively. With longer epinephrine exposure, the increase in cell shortening continuously declined to 134.6+/-7.9% with no change in FURA-2 fluorescence. Acute administration of T3 after epinephrine exposure lowered contractile performance from 136.2 +/- 15.5% to 102 +/- 8.2 %, after 10 minutes. In control experiments acute administration of T3 increased basal contractile response from 100% to 115.8+/-3.3% (p<0.01; n = 8; 5 min). This study confirms previous reports of impaired cardiac function after high catecholamine exposure. High catecholamine exposure is associated with a desensitization of contractile proteins for calcium. Acute T3 administration increased contractility in untreated myocytes, but further depressed myocyte shortening in epinephrine-treated cells. Our results show that T3 is ineffective at restoring myocardial contractility after excessive catecholamine stimulation.

Effects of cytosolic ATP on spontaneous and triggered Ca2+-induced Ca2+ release in permeabilised rat ventricular myocytes.

1. The effects of cytosolic ATP on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin-permeabilised rat ventricular myocytes. [Ca2+] within the cells was monitored using Fura-2 or Fluo-3 fluorescence. Spontaneous cyclic Ca2+ release from the SR was induced by increasing the bathing [Ca2+] to 200-300 nM, in solutions weakly Ca2+ buffered with 0.05 mM EGTA. Alternatively, Ca2+-induced Ca2+ release (CICR) was triggered by a rapid increase in [Ca2+] induced by flash photolysis of Nitr-5 (0.08 mM), replacing EGTA in the solution. 2. Stepwise reductions in [ATP] were associated with corresponding decreases in the frequency and increases in the amplitude of spontaneous Ca2+ transients. A decrease from 5 mM to 0. 1 mM ATP, reduced the release frequency by 48.6 +/- 7 % (n = 7) and almost doubled the amplitude of the Ca2+ transient. Marked prolongation of the spontaneous Ca2+ transient occurred when [ATP] was further reduced to 10 microM, consistent with inhibition of the SR Ca2+ pump. 3. These effects of ATP were compared with other interventions that inhibit Ca2+ uptake or reduce the sensitivity of the SR Ca2+ release mechanism. Inhibition of the SR Ca2+ pump with cyclopiazonic acid (CPA) markedly reduced the spontaneous Ca2+ release frequency, without changing the amplitude. The descending phase of the Ca2+ transient was prolonged in the presence of CPA, while the rising phase was unaffected. In contrast, desensitisation of the SR Ca2+ release mechanism with tetracaine decreased the frequency of spontaneous release, but markedly increased the amplitude. 4. CICR triggered by flash photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP] than spontaneous release and was generally delayed by a decrease to 2.5 mM ATP. In the presence of 0.1-0.2 mM ATP, release often failed completely or was not consistently triggered. Some preparations exhibited Ca2+ release 'alternans', whereby every alternate trigger induced a response. 5. These results suggest that the increase in spontaneous Ca2+ release amplitude and the decrease in frequency that occurs as [ATP] is reduced from 1 mM to 100 microM, is mainly due to desensitisation of the SR Ca2+ release mechanism, which allows the SR Ca2+ content to reach a higher level before release occurs. At very low [ATP], a reduction in the SR Ca2+ uptake rate may also contribute to the decrease in release frequency. CICR triggered by photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP]. The possible underlying mechanisms and the relevance of these results to myocardial ischaemia or hypoxia is considered.

Tyrosine kinase inhibitors and Ca 2+ signaling: direct interactions with fura-2

Selective inhibitors were used to study the role of tyrosine kinases in α 1A-adrenoceptor-mediated responses in transfected PC12 cells. Ca 2+ responses to noradrenaline were measured using fura-2, and the effects of genistein, tyrphostin A25, and herbimycin A were examined. Neither genistein nor herbimycin A pretreatment altered noradrenaline-induced Ca 2+ responses, although tyrphostin A25 pretreatment caused some reduction. However, acute addition of genistein quickly reversed the apparent noradrenaline response, apparently, through a direct interaction with cytoplasmic fura-2. Both genistein and tyrphostin A25, at concentrations similar to those used to inhibit tyrosine kinases, markedly reduced fluorescence of fura-2 excited by both 340 and 380 nm, and genistein also reduced the 340/380 ratio. Tyrosine kinase inhibitors did not alter noradrenaline stimulated inositol phosphate formation in α 1A-PC12 cells. These results suggest that tyrosine kinases are not involved in second messenger responses to α 1A-adrenoceptors, but that tyrosine kinase inhibitors can interact directly with fura-2.

Fendiline increases [Ca 2+] i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca 2+ followed by capacitative Ca 2+ entry

The effect of fendiline, a documented inhibitor of L-type Ca 2+ channels and calmodulin, on Ca 2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca 2+ probe. Fendiline at 5–100 μM significantly increased [Ca 2+] i concentration-dependently. The [Ca 2+] i rise consisted of an initial rise and a slow decay. External Ca 2+ removal partly inhibited the Ca 2+ signals induced by 25–100 μM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca 2+ influx and internal Ca 2+ release. In Ca 2+-free medium, pretreatment with 50 μM fendiline nearly abolished the [Ca 2+] i rise induced by 1 μM-thapsigargin, an endoplasmic reticulum Ca 2+ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca 2+. This indicates that the internal Ca 2+ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 μM, fendiline caused Mn 2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La 3+ by 50%, implying that fendiline-induced Ca 2+ influx has two components separable by La 3+. Consistently, 0.1 mM La 3+ pretreatment suppressed fendiline-induced [Ca 2+] i rise, and adding La 3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca 2+ increased [Ca 2+] i after preincubation with 50–100 μM fendiline in Ca 2+-free medium. However, 50–100 μM fendiline inhibited 1 μM thapsigargin-induced capacitative Ca 2+ entry. Pretreatment with 40 μM aristolochic acid to inhibit phospholipase A 2 inhibited 50 μM fendiline-induced internal Ca 2+ release by 48%, but inhibition of phospholipase C with 2 μM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca 2+] i in MDCK cells by releasing internal Ca 2+ in a manner independent of inositol-l,4,5-trisphosphate (IP 3), followed by external Ca 2+ influx.

Permeation and Gating Properties of the Novel Epithelial Ca2+ Channel

The recently cloned epithelial Ca(2+) channel (ECaC) constitutes the Ca(2+) influx pathway in 1,25-dihydroxyvitamin D(3)-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca(2+) concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca(2+) gradient, demonstrating the distinctive Ca(2+) permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below -40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba(2+) or Sr(2+) replaced Ca(2+) and was absent in Ca(2+)-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P(Ca):P(Na) calculated from the reversal potential at 30 mM [Ca(2+)](o) was larger than 100. The divalent cation selectivity profile is Ca(2+) > Mn(2+) > Ba(2+) approximately Sr(2+). Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca(2+) was replaced by Ba(2+) and was virtually abolished if [Ca(2+)](o) was lowered to 1 nM. In conclusion, ECaC is a Ca(2+) selective channel, exhibiting Ca(2+)-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.