Flufenamate and Gd3+ inhibit stimulated Ca2+ influx in the epithelial cell line CFPAC-1

The relevant influx pathway for stimulated Ca²⁺ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd³⁺, both known pharmacological blockers of non-selective cation currents in other epithelial preparations, we tested whether the stimulated Ca²⁺ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca²⁺ influx into CFPAC-1 cells was stimulated by either ATP (10^–4 and 10^–5 mol/l), carbachol (CCH, 10^–4 mol/l) or thapsigargin (TG, 10^–8 mol/l). Three different experimental approaches were used. (1) Because the plateau phase of an agonist-induced [Ca²⁺]_i transient reflects Ca²⁺ influx into these cells, we investigated the influence of Flu and Gd³⁺ on the level of the stimulated [Ca²⁺]_i plateau. (2) The fura-2 Mn²⁺-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the refilling period after agonist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd³⁺ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd³⁺ were potent inhibitors of the stimulated Ca²⁺ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca²⁺]_i plateau in a concentration dependent manner, with an IC₅₀ value of 33 mol/l (n = 6). Similar results were obtained for the CCH-(n = 5) and the TG-induced (n = 5) [Ca²⁺]_i plateau. Gd³⁺ concentration dependently inhibited the stimulated Ca²⁺ plateau. A complete block of the ATP-induced [Ca²⁺]_i plateau was seen at 0.5 mol/l (ATP 10^–5 mol/l, n = 8). The second approach showed that Flu (10^–4 mol/l) completely inhibited the ATP- (10^–5 mol/l, n = 3), CCH-(10^–4 mol/l, n = 4) and TG-(10^–8 mol/l, n = 3)-induced fura-2 Mn²⁺ quench. Gd³⁺ also inhibited the fura-2 Mn²⁺-quenching rate (n = 9). The third approach showed that Flu (n = 6) and Gd³⁺ (n = 8) inhibited the refilling of the ATP-sensitive intracellular Ca²⁺ store. These results show that inhibitors of non-selective cation currents in other epithelial preparations are potent inhibitors of stimulated Ca²⁺ influx in CFPAC-1 cells. Whether this inhibitory effect concerns a non-selective cation channel remains to be established.     

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca²⁺ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10^–5 mol/l)-induced intracellular Ca²⁺ transient, (2) a reduced fura-2 Mn²⁺-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca²⁺ as a function of membrane voltage, we measured simultaneously the intracellular Ca²⁺ activity ([Ca²⁺]_i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT₂₉ cells. Ca²⁺ influx was either stimulated by ATP (10^–5 mol/l) or thapsigargin (TG, 10^–8 mol/l). After [Ca²⁺]_i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca²⁺]_i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca²⁺]_i (ATP: 27.5±10 nmol/l per 10 mV, n=6; TG: 19 ±7.9 nmol/l per 10 mV, n=12). The summarised data show a linear relationship between the fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca²⁺]_i (n=9). Under extracellular Ca²⁺-free conditions [Ca²⁺]_i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca²⁺ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca²⁺]_i. This may be due either to a change of driving force for Ca²⁺ into the cell, or may reflect voltage-dependent regulation of the respective Ca²⁺ entry mechanism.     

Desensitization of the bradykinin-induced rise in intracellular free calcium in cultured endothelial cells

We studied the cellular mechanism involved in the desensitization of cultured endothelial cells to bradykinin. Bradykinin (10 nmol/l) evoked a rise in the intracellular free calcium concentration ([Ca _i ²⁺ ]), measured with the fluorescent probe indo-1, from 137±30 (±SEM) to 623±101 nmol/l. Cells were desensitized to bradykinin by repetitive stimulation with the peptide over 10 min, after which they no longer responded to bradykinin. However, purinergic stimulation with ATP (10 mol/l) elicited the same increase in [Ca _i ²⁺ ] in endothelial cells desensitized to bradykinin as in cells never exposed to bradykinin. The initial peak of [Ca _i ²⁺ ] after stimulation with bradykinin or ATP was not affected by removal of extracellular calcium ions, indicating mobilization of Ca²⁺ from intracellular stores. Since GTP-binding proteins (G-proteins) are probably involved in the receptor-mediated stimulation of endothelial cells, we also tested the effects of sodium fluoride (NaF), a reported direct stimulator of G-proteins, on endothelial [Ca _i ²⁺ ]. NaF (5 mmol/l) increased [Ca _i ²⁺ ] to 412±88 nmol/l in control cells and was equally effective in cells desensitized to bradykinin. We conclude that the homologous desensitization to bradykinin does not occur at the level of intracellular signal transduction but at the level of membrane receptors.     

Diethylstilbestrol increases intracellular calcium in lens epithelial cells

The effects of diethylstilbestrol (DES) on steady-state intracellular calcium concentration ([Ca²⁺]_i) and resting Ca²⁺ influx were examined in primary cultures of bovine lens epithelial cells using conventional fluorometric techniques (Fura-2). At low concentrations (10 M), DES usually induced relatively rapid increases in [Ca²⁺]_i that occurred over an interval of 10–50 s and that persisted for several minutes in the continued presence of the drug. In about 10% of the cells, cyclic oscillations in [Ca²⁺]_i were seen after adding 10 M DES. At higher concentrations (100 M), the drug induced more prolonged increases in [Ca²⁺]_i lasting several minutes. DES did not affect Mn²⁺ quench determinations of resting Ca²⁺ influx, and neither 100 M GdCl₃, which blocked resting Ca²⁺ influx, nor low [Ca²⁺]_o solutions substantially diminished the influence of DES on [Ca²⁺]_i. Pretreatment of cells with the smooth endoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibitors cyclopiazonic acid (CPA) or thapsigargin completely abolished the effect of 10 M DES on [Ca²⁺]_i, while the IP₃ receptor blocker 2-aminoethoxydiphenyl borane (2-APB) had no effect. These results indicate that DES releases CPA-sensitive stores of intracellular Ca²⁺, perhaps by inhibiting SERCA-dependent Ca²⁺ sequestration.     

Hormone-mediated Ca2+ transients in isolated renal cortical thick ascending limb cells

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca²⁺ ([Ca²⁺]_i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca²⁺]_i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca²⁺]_i in a dose-dependent manner. [Ca²⁺]_i maximally increased from 85±5 nmol/l to 608±99 nmol/l with PTH, 10^–6M, and to 766±162 nmol/l with AVP, 10^–7 M. The increment in [Ca²⁺]_i by both hormones was by intracellular Ca²⁺ release and entry through plasma membrane Ca²⁺ channels. 8-Bromoadenosine-3, 5-cyclic monophosphate (8-BrcAMP), 10^–4M, increased [Ca²⁺]_i(basal 83±3 to 427±121 nmol/l) but only from internal sources as nifedipine (10 mol), ([Ca²⁺]_i changes: 86±4 to 390±29 nmol/l) and removal of bath Ca _o ²⁺ , ([Ca²⁺]_ichanges: 84±6 to 517±142 nmol/l), were without effect on agonist-induced [Ca²⁺]_i. Thapsigargin, 1.5 mol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca²⁺ transients, and partially inhibited PTH-mediated Ca²⁺ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca²⁺ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca²⁺]_i transients. The responses partially returned following down-regulation of PKC with prolonged exposure to phorbol esters. These data suggest that PKC activation modulates agonist-induced Ca²⁺ release and entry, possibly through actions on intracellular release mechanisms. In summary, PTH and AVP stimulate Ca²⁺ signals by similar pathways involving cAMP and inositol 1,3,4-trisphosphate activity at similar sites on the endoplasmic reticulum and plasma membrane. These results suggest that peptide hormones may act through Ca²⁺ and be modulated by different pathways which may have diverse effects on cTAL function.     

A method for recording intracellular [Ca2+] transients in cardiac myocytes using calcium green-2

Calcium green-2 (Ca green) is a non-ratio-metric fluorescent Ca²⁺ indicator with an affinity for Ca²⁺ (dissociation constant K_d=3 M) that is lower than more commonly used indicators such as fura-2 and fluo-3. This low Ca²⁺ affinity, coupled with a high quantum yield, allows cells to be loaded with low concentrations of Ca green, avoiding problems of cytosolic Ca²⁺ buffering and a low signal-to-noise ratio. This communication presents a method for monitoring intracellular [Ca²⁺] changes in isolated rat ventricular myocytes loaded with Ca green and the fluorescent pH indicator carboxy SNARF-1 (SNARF). SNARF provides a Ca²⁺-insensitive signal with which Ca green fluorescence can be corrected for cell motion and dye-loading artifacts.     

Effect of ATP,carbachol and other agonists on intracellular calcium activity and membrane voltage of pancreatic ducts

The pancreatic duct has been regarded as a typical cAMP-regulated epithelium, and our knowledge about its Ca²⁺ homeostasis is limited. Hence, we studied the regulation of intracellular calcium, [Ca²⁺]_i, in perfused rat pancreatic ducts using the Ca²⁺-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, V _bl, of individual cells. The resting basal [Ca²⁺]_i was relatively high, corresponding to 263±28 nmol/l, and it decreased rapidly to 106±28 nmol/l after removal of Ca²⁺ from the bathing medium (n=31). Carbachol increased [Ca²⁺]_i in a concentration-dependent manner. At 10 mol/l the fura-2 fluorescence ratio increased by 0.49±0.06 (n=24), corresponding to an increase in [Ca²⁺]_i by 111±15 nmol/l (n=17). ATP, added to the basolateral side at 0.1 mmol/l and 1 mmol/l, increased the fluorescence ratio by 0.67±0.06 and 1.01±14 (n=46; 12), corresponding to a [Ca²⁺]_i increase of 136±22 nmol/l and 294±73 nmol/l respectively (n= 15; 10). Microelectrode measurements showed that ATP (0.1 mmol/l) hyperpolarized V _bl from –62±3 mV to-70±3 mV, an effect which was in some cases only transient (n=7). This effect of ATP was different from that of carbachol, which depolarized V_bl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of V _bl (n=4). Several other putative agonists of pancreatic HCO ₃ ^– secretion were also tested for their effects on [Ca²⁺]_i. Bombesin (10 nmol/l) increased the fura-2 fluorescence ratio by 0.24±0.04 (n=8), neurotensin (10 nmol/l) by 0.25±0.04 (n=6), substance P (0.1 mol/l) by 0.22±0.06 (n=6), and cholecystokinin (10 nmol/l) by 0.14±0.03 (n=7). Taken together, our studies show that Ca²⁺ homeostasis plays a role in pancreatic ducts. The most important finding is that carbachol and ATP markedly increase [Ca²⁺]_i, but their different electrophysiological responses indicate that intracellular signalling pathways may differ.Preliminary reports of the present study have been presented at the 72nd Meeting of the German Physiological Society, March 1993     

Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca²⁺, 37°C, pH 7.4), 10%–20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca²⁺]_i) of these cells was 106±4nM versus 77±3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity 50 m/s, maximal Ca amplitude=195±11nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca²⁺]_i increase induced by K⁺ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca²⁺]_i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).     

Spatial and temporal control of intracellular free Ca2+ in chick sensory neurons

Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca²⁺ concentration, [Ca]_i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]_i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]_i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]_i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]_i. During wash-out [Ca]_i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]_i was quickly restored. [Ca]_i recovery was delayed after treating the cell with 2 M thapsigargin, an inhibitor of the Ca²⁺ pump of internal Ca²⁺ stores. Caffeine (10 mM) transiently increased [Ca]_i. A second caffeine application produced smaller [Ca]_i changes due to the prior depletion of Ca²⁺ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 M) transiently increased [Ca]_i both in the standard and Ca²⁺-free solution. [Ca]_i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]_i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La³⁺ and in Ca²⁺-free solutions, but which was greatly diminished in Na⁺-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]_i transients and in the inward current induction.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

The rate of tetanic relaxation is correlated with the density of calcium ATPase in the terminal cisternae of thyrotoxic skeletal muscle

We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patchclamped cells, as well as cytosolic calcium ([Ca²⁺]_i) in fura-2-loaded cell suspensions and monolayers. Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels. When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant. With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV. Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of 1 M. Isoproterenol (1 M.) or forskolin (10 M) increased currents in approximately half of the cells tested. Forskolin (10 M) increased single-channel activity in five of eight cell-attached patches. After cells had been quiescent for several weeks, cell suspensions showed changes in resting [Ca²⁺]_i in response to DHPs and increased potassium. Most confluent monolayers of cells showed spontaneous transient elevations in [Ca²⁺]_i. Bath application of Bay K 8644 increased the frequency and magnitude of these [Ca²⁺]_i transients, whereas nifedipine abolished the transients. These data suggest that the [Ca²⁺]_i transients were due to synchronous action potentials in electrically coupled cell monolayers.     

Effects of caffeine on intracellular calcium,calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells

Caffeine elicits physiological responses in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular organelles. Here we investigate the effects of caffeine on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ionic currents in anterior pituitary cells (GH₃) cells. Caffeine has a biphasic effect on Ca²⁺-activated K⁺ current [I _K(Ca)]: it induces a transient increase superimposed upon a sustained inhibition. While the transient increase coincides with a rise in [Ca²⁺]_i, the sustained inhibition of I _K(Ca) is correlated with a sustained inhibition of the L-type Ca²⁺ current. The L-type Ca²⁺ current is also inhibited by other agents that mobilize intracellular Ca²⁺, including thyrotropin releasing hormone (TRH) and ryanodine, but in a matter distinct from caffeine. Unlike the caffeine effect, the TRH-induced inhibition washes-out under whole-cell patch-clamp conditions and is eliminated by intracellular Ca²⁺ chelators. Likewise, the ryanodine-induced inhibition desensitizes while the caffeine-induced inhibition does not. Simultaneous [Ca²⁺]_i and Ca²⁺ current measurements show that caffeine can inhibit Ca²⁺ current without changing [Ca²⁺]_i. Single-channel recordings show that caffeine reduces mean open time without affecting single-channel conductance of L-type channels. Hence the effects of caffeine on ion channels in GH₃ cells are attributable both to mobilization of intracellular Ca²⁺ and to a direct effect on the gating of L-type Ca²⁺ channels.     

Effects of diadenosine polyphosphates,ATP and angiotensin II on cytosolic Ca2+ activity and contraction of rat mesangial cells

Diadenosine polyphosphates (Ap _n A) are known to influence cellular Ca²⁺ activity ([Ca²⁺]_i) in several cells. Their vasoactive potency has been described in various systems including the kidney. We examined the effects of diadenosine polyphosphates, adenosine 5-triphosphate (ATP) and angiotensin II (Ang II) on cytosolic Ca²⁺ activity of mesangial cells (MC) in culture obtained from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. [Ca²⁺]_i was measured as a fluorescence ratio F ₃₄₀/F ₃₈₀ with the fura-2 technique using three excitation wavelengths (340 nm, 360 nm and 380 nm) and a photon counting tube. Resting [Ca²⁺]_i was not significantly different in MC from WKY and SHR rats and was measured as 132±9 nmol/l (n=65) and 114±12 nmol/l (n=36), respectively. Diadenosine polyphosphates (Ap₃A–Ap₆A) increased [Ca²⁺]_i transiently with an initial peak and a secondary plateau phase comparable to the effects of ATP or Ang II. Increases in [Ca²⁺]_i induced by all these agonists were not significantly different between MC of WKY and SHR rats. ATP, Ap₃A, Ap₄A, Ap₅A, Ap₆A (each 5 mol/l) increased the fura-2 fluorescence ratio initially by 0.66±0.09 (n=33), 0.52±0.08 (n=18), 0.25±0.05 (n=16), 0.09±0.06 (n=7), 0.09±0.04 (n=11), respectively. A half-maximal initial increase in the fura-2 fluorescence ratio was reached at 22 nmol/l, 0.9 mol/l, 2.0 mol/l and 4.0 mol/l with Ang II, Ap₃A, ATP and Ap₄A, respectively. Ap₄A (100 mol/l, n=18) led to a reversible contraction of MC. Diadenosine polyphosphates increase [Ca²⁺]_i in rat MC, in a similar manner to ATP or Ang II and lead to a contraction of MC, suggesting that these nucleotides are also involved in the control of glomerular haemodynamics.     

Potentiating and depressant effects of metabotropic glutamate receptor agonists on high-voltage-activated calcium currents in cultured retinal ganglion neurons from postnatal mice

This study was aimed at clarifying the role of metabotropic glutamate receptors (mGluRs) in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in postnatal mouse retinal ganglion neurons (RGNs). RGNs were maintained for 1–2 weeks in vitro by adding brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) to the culture medium. In order to select these cells for electrophysiological measurements, RGNs were vitally labelled with an antibody against Thy-1.2. Voltage-activated Ca²⁺ currents [I _Ca(V)] were recorded with patch electrodes in the wholecell configuration. It was found that racemic ±-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD) or its active enantiomer 1S,3R-ACPD rapidly and reversibly either enhanced or depressed I _Ca(V). Quisqualate (QA), l-2-amino-4-phosphonobutyrate (l-AP4) and the endogenous transmitter glutamate induced similar effects when ionotropic glutamate receptors were blocked with d-2-amino-5-phosphonovalerate (d-APV) and 6,7-dinitroquinoxaline-2, 3-dione (DNQX). - Conotoxin GVIA (-CgTx GVIA), but not nifedipine prevented modulation of I _Ca(V) by mGluR agonists. The depression of I _Ca(V) by t-ACPD was irreversible when cells were dialysed with guanosine-5-O-(3-thiotriphosphate) (GTP[-S]). Ratio measurements of fura-2 fluorescence in Thy-1+ cells showed that neither t-ACPD, QA nor l-AP4 affected [Ca²⁺]_i by liberation of Ca²⁺ from intracellular stores. Our results suggest that cultured RGNs express mGluRs. These receptors cannot induce Ca²⁺ release from intracellular stores but regulate [Ca²⁺]_i by a fast and reversible, G-protein-mediated action on a subpopulation of voltage-activated Ca²⁺ channels.     

Extracellular Ca2+-dependent and independent calcium transient in fetal myotubes

Spatio-temporal changes in the intracellular calcium concentration [Ca²⁺]_i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca²⁺ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca²⁺]_i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 M lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca²⁺]_i by stimulating Ca²⁺ release from the sarcoplasmic reticulum independently of extracellular Ca²⁺ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca²⁺ entry through calcium channels was suppressed.     

Intracellular calcium responses and shape conversions induced by endothelin in cultured subepithelial fibroblasts of rat duodenal villi

Subepithelial fibroblasts of rat duodenal villi were cultured and the physiological characteristics were studied using fura-2 fluorescence. The intracellular calcium concentration (Ca _i ²⁺ ) responded to various substances, i.e., endothelins (ET1 and ET3), substance P, serotonin, angiotensin II, ATP, and bradykinin. The Ca _i ²⁺ responses to ET1 (>0.1 nM) and ET3 (>1 nM) were transient and sometimes followed oscillations that consisted of an initial Ca²⁺ release from the intracellular store and a sustained Ca²⁺ influx. Simultaneously with Ca _i ²⁺ measurement, changes in the cell shape were monitored using fluorescence intensity upon 360-nm excitation. Stellate cells (with thick cell body and slender processes), formed as a result of 1 mM dibutyryl(Bt₂)-cAMP treatment, began to change immediately after the short-term application of the endothelin and became flat about 20 min later. This process was not affected by the depletion of extracellular Ca²⁺ or by the treatment with BAPTA acetoxymethyl ester that completely suppressed the Ca _i ²⁺ response. Substance P (>100 nM) increased Ca _i ²⁺ , but did not induce any morphological changes. The conversion of the shape from flat to stellate, induced by Bt₂cAMP treatment, was not accompanied by any Ca _i ²⁺ change. BQ-123, a specific blocker of the ET_A-type receptor, did not block either Ca _i ²⁺ change or shape conversion at low (100 nM) concentration. The results indicated that shape conversion in subepithelial fibroblasts did not require any Ca _i ²⁺ response. Our findings regarding the characteristics of subepithelial fibroblasts in intestinal villi imply a functional similarity to astrocytes in the brain.     

Effects of substrate-free anoxia and veratridine on intracellular calcium concentration in isolated rat ventricular cardiomyocytes

Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured in freshly isolated rat ventricular cardiomyocytes during substrate-free anoxia. Cardiomyocytes were loaded with fura-2 and incubated in an anoxic chamber in which a pO₂ equal to 0 mmHg was realized by inclusion of Oxyrase. [Ca²⁺]_i was measured in individual cells using digital imaging fluorescence microscopy. During anoxia, the shape of cardiomyocytes changed from a relaxed-elongated form into a rigor configuration within 15 min after the onset of anoxia. After the cells had developed the rigor state, a delayed rise in [Ca²⁺]_i reached a stable maximal level within 45 min. The mean values for the pre-anoxic and maximal anoxic [Ca²⁺ _i were 52±3 nM (N=42) and 2115±59 nM (N=45), respectively. The purported Na⁺ overload blocker R 56865, significantly reduced maximal anoxic [Ca²⁺]_i to 553±56 nM (P<0.05), implicating a role of elevated intracellular Na⁺ in the anoxia-induced increase in [Ca²⁺]_i. Veratridine (30 M), which induces Na⁺ overload, increased [Ca²⁺]_i to 787±39 nM. The compound R 56865 reduced veratridine-induced increases in [Ca²⁺]_i to 152±38 nM. Upon reperfusion, after 45 min of anoxia, two distinct responses were observed. Most often, [Ca²⁺]_i decreased upon reperfusion without a change in morphology or viability, while in the minority of cases, [Ca²⁺]_i increased further followed by hypercontraction and loss of cell viability. The mean value for [Ca²⁺]_i 10 min after reperfusion of the former group, was 752±46 nM (N=38). The cardiomyocyte cell shape could be followed by monitoring changes in the total fura-2 fluorescence (340+380 nm signal). Within 15 min after the onset of anoxia, the total fluorescence signal increased suddenly, before [Ca²⁺]_i started to rise, coinciding with the onset of rigor contraction induced by ATP depletion.     

Activation of calcium influx by ATP and store depletion in primary cultures of renal proximal cells

Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 M) induced a biphasic increase of [Ca²⁺]_i measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca²⁺]_i from 106±11 nM (n=36) to 1059±115% (n=29) of the resting level within 10 s. In the presence of external calcium, [Ca²⁺]_i then decreased within 3 min to a sustained level (398±38%,n=8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca²⁺ uptake, blocked by lanthanum (10 M) and verapamil (100 M) but not by the nifedipin (25 M). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipininsensitive calcium channels, located on the apical membrane of the cells. As indicated by⁸⁶Rb⁺ efflux measurements, ATP activated a potassium efflux that was blocked by barium andLeiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca²⁺-sensitive K⁺ channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATPevoked hyperpolarization of the plasma membrane leads to increased Ca²⁺ influx, which facilitates the replenishment of the internal stores.     

Intracellular free calcium concentration / force relationship in rabbit inferior vena cava activated by norepinephrine and high K+

The changes in isometric force and the underlying fluctuations in intracellular free calcium concentration ([Ca²⁺]_i) were monitored simultaneously in thin sheets of rabbit inferior vena cava loaded with the fluorescent Ca²⁺ indicator fura-2. In resting tissues bathed in physiological saline solution, the estimated [Ca²⁺]_i was approximately 105 nM. The -adrenergic agonist norepinephrine (10 M) caused an initial rise in [Ca²⁺]_i to 264 nM during force development, which dropped to 216 nM during force maintenance. The maintained norepinephrine-induced increase in force and [Ca²⁺]_i was reversed in Ca²⁺-free (2 mM EGTA) solution. Membrane depolarization by high K⁺ (80 mM) significantly increased [Ca²⁺]_i to 234 nM. Compared to norepinephrine, high K⁺ caused about the same steady-state increase in [Ca²⁺]_i, but a smaller increase in force. [Ca²⁺]_i/force curves were constructed at different concentrations of extracellular Ca²⁺, with either norepinephrine or high K⁺ as a stimulant. The curve generated with norepinephrine was located to the left of that generated with high K⁺.