Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5–15 min) between spontaneous contractions [Ca²⁺]_i continues to decrease after full mechanical relaxation to reach a minimal level of 86±8 nM (n=9) just before the start of the next contraction. During the spontaneous contractions (5–15 min) [Ca²⁺]_i reached a maximum of 211±19 nM and then oscillated between 155±16 nM and 194±9 nM. Increased extracellular Ca²⁺ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca²⁺]_i both in the relaxed and contracted states. In 10 mM extracellular Ca²⁺, addition of AlF₄ ^–, as 1 mM NaF + 10 M AlCl₃, caused a sustained increase in [Ca²⁺]_i and maintained force. Addition of verapamil (10 M) in this situation decreased [Ca²⁺]_i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca²⁺]_i, possibly via inactivation of Ca²⁺-dependent K⁺ channels.     

Propagation of excitation-contraction coupling into ventricular myocytes

This paper examines the [Ca²⁺]_i transient in isolated rat heart cells using a laser scanning confocal microscope and the calcium indicator fluo-3. We find that the depolarization-evoked [Ca²⁺]_i transient is activated synchronously near the surface and in the middle of the heart cell with similar kinetics of activation. The time of rise of the transient did not depend on whether the sarcoplasmic reticulum (SR) Ca-release was abolished (by thapsigargin and ryanodine). The synchrony of activation and the similarity of levels of [Ca²⁺]_i at the peripheral and deeper myoplasm (regardless of the availability of SR Ca-release) shows that sarcolemmal Ca channels and SR Ca-release channels are distributed throughout the rat heart cell and that the propagation of the action potential into the interior of the cell is rapid. In addition, the activation of calcium release from the SR by CICR is rapid (2 ms) when compared to the time-course of calcium influx via the sarcolemmal Ca channel.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

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.     

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery

To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca²⁺ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca²⁺ mobilization were investigated by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using Fura 2 in Ca²⁺-containing or Ca²⁺-free solution. The resting [Ca²⁺]_i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca²⁺, 10 M ACh or 128 mM K⁺ produced a phasic, followed by a tonic, increase in [Ca²⁺]_i but 20 mM caffeine produced only a phasic increase. In Ca²⁺-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca²⁺]_i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K⁺) transiently increased [Ca²⁺]_i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca²⁺]_i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca²⁺. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca²⁺]_i induced by 128 mM K⁺ and also by 10 M ACh in Ca²⁺-containing solution. In Ca²⁺-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca²⁺]_i following application of 20 mM caffeine. The level of [Ca²⁺]_i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca²⁺-free solution. These results suggest that both ACh and caffeine release Ca²⁺ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca²⁺]_i and inhibited the rate of decline of [Ca²⁺]_i following wasthout of high K⁺ or ACh in Ca²⁺-containing solution suggests that SR may negatively regulate the resting [Ca²⁺]_i in smooth muscle cells of the porcine coronary artery.     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

Intracellular Ca2+ concentrations are not elevated in resting cultured muscle from Duchenne (DMD) patients and in MDX mouse muscle fibres

The free intracellular calcium concentration, [Ca²⁺]_i, was studied in single myotubes using the fluorescent Ca²⁺ indicator fura-2. Myotubes cultured from satellite cells of small muscle specimens from Duchenne muscular dystrophy (DMD) patients were compared with human control myotubes and with myotubes cultured from MDX and control mouse muscle satellite cells. The resting [Ca²⁺]_i levels in DMD and control myotubes were not significantly different, i. e. 104 ±26 nM (mean ± SD, n=190 cells from eight DMD patients) compared with 97±25 nM (175/seven controls) and were not significantly lower than the corresponding murine values (154±33 nM, n=135 MDX myotubes; 159±34 nM, n=135 controls). All myotubes reacted to 10 M acetylcholine or 40 mM KCl with fast transient increases of [Ca²⁺]_i. After application of a hyposmotic (130 mOsm) solution, [Ca²⁺]_i was increased 1.5- to 3-fold within 2–3 min, the DMD myotubes tending to stronger reactions (significantly higher [Ca²⁺]_i in 2 out of 6 cases). The response was usually transient, [Ca²⁺]_i decreasing to the initial level within 10 min. Gadolinium (50 M) reduced the response by 50%–70%, indicating that the osmotic shock increased Ca²⁺ influx. During exposure to high (15 mM) [Ca²⁺]e, [Ca²⁺]_i of DMD and control cells was 1.5- to 2-fold higher. Adult muscle fibres from MDX mice and controls showed identical Ca²⁺ resting levels (n=45 fibres from three mice in each case), but did not respond to decreased external osmolarity with a change in [Ca²⁺]_i. The results indicate that lack of dystrophin in muscle fibres does not necessarily lead to increased [Ca²⁺]_i. It is suggested that increased [Ca²⁺]_i is probably a secondary consequence of fibre damage.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Thapsigargin discharges intracellular calcium stores and induces transmembrane currents in human endothelial cells

We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase), on membrane currents and on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca²⁺]_i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 mol/l induced a slow increase in [Ca²⁺]_i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca²⁺]_i depended on the extracellular Ca²⁺ concentration ([Ca²⁺]_o: it declined after removal of extracellular Ca²⁺, but increased again when [Ca²⁺]_o was augmented, indicating that the response depends on a transmembrane influx of Ca²⁺ ions. The peak amplitude of the histamine-induced Ca²⁺ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca²⁺]_i induced by thapsigargin than during the rising phase of the changes in [Ca²⁺]_i. The decline of the Ca²⁺ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of –40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca²⁺ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca²⁺ pump in endothelial cells increases [Ca²⁺]_i. The tonic component of this increase might be partly due to opening of non-selective Ca²⁺-permeable cation channels activated by depletion of intracellular stores.     

Effects of 5-hydroxytryptamine (serotonin) and forskolin on intracellular free calcium in isolated and fura-2 loaded smooth-muscle cells from the anterior byssur retractor (catch) muscle ofMytilus edulis

Effects of 5-hydroxytryptamine (5-HT) and forskolin on intracellular free calcium concentration ([Ca²⁺]_i) were studied in suspensions of fura-2 loaded smooth-muscle cells from the anterior byssus retractor catch muscle ofMytilus edulis. The successive addition of 5 mM carbachol (CCh) and 100 mM KCl to the suspension evoked a transient elevation of [Ca²⁺]_i from the resting value of 124±2.7 nM (mean ± SE,n=18) to 300–400 nM, which was associated with contraction. The change in [Ca²⁺]_i induced CCh was concentration-dependent with the EC₅₀ of 10^–5 M. The resting [Ca²⁺]_i was unaffected by 10 M 5-HT. The change in [Ca²⁺]_i induced by 5 mM CCh was suppressed by 5-HT from 167±14.0 (n=11) to 124±14.9 (n=8) nM whereas that induced by 100 mM KCl was enhanced from 321±31.9 to 405±17.6 nM (n=8). 5-HT applied during the decaying phase of the CCh response caused a rapid decline in [Ca²⁺]_i. In both the responses to CCh and KCl, the falling phase was accelerated by 5-HT. 10 M forskolin, a potent activator of adenylate cyclase, mimicked the effects of 5-HT as did a membrane-permeant cyclic AMP analogue, 8-parachlorophenylthio cyclic AMP (cpt-cAMP). Application of 100 M cpt-cAMP partially suppressed the Ca²⁺ _i response to CCh and enhanced that to KCl.d-Tubocurarine (500 M) added during the decaying phase of the response induced by 100 M CCh, caused a rapid decline in [Ca²⁺]_i similar to that caused by both 5-HT and forskolin. In essentially Ca²⁺-free sea water, or in the presence of 10 M D600 in seawater containing 4 mM, Ca²⁺, the response to CCh was partially suppressed, whereas that to KCl was completely abolished, demonstrating a CCh-induced release of intracellularly stored Ca²⁺. The remaining component of the response to CCh, in either Ca²⁺-free sea water or in the presence of D600, was abolished by both 5-HT and forskolin. The results suggest that 5-HT has multiple effects on [Ca²⁺]_i in the ABRM, and implicate cyclic AMP in this effect, and that one of the mechanisms underlying these responses is the inhibition of an agonist-induced release of stored Ca²⁺. In addition, that Ca²⁺ _i is at, or close to resting values during the catch state.     

Effect of BAY K8644 on cytosolic calcium transients and contraction in embryonic cardiac ventricular myocytes

Cytosolic calcium transients were recorded from spontaneously beating chick embryonic myocardial cell aggregates loaded with the fluorescent [Ca²⁺]_i indicator, indo-1. Calcium transients rose rapidly from an end-diastolic [Ca²⁺]_i of 230±18 nM to a peak systolic [Ca²⁺]_i of 619±34 nM (n=21). Relaxation of the transients was slow, and continued throughout diastole. Bay K8644 (0.5 M) markedly prolonged the action potential and caused similar prolongation of the calcium transients. Calcium transients in the presence of Bay K8644 had an inflection on their rising phase, which was followed by a more gradual increase that continued until the membrane had repolarized to a negative potential of –15 to –30 mV. Bay K8644 caused marked elevation of peak systolic [Ca²⁺]_i to 955±56 nM (P<0.002), with concomitant elevation of end-diastolic [Ca²⁺]_i to 400±36 nM (P<0.002). Optical recordings of contraction showed changes similar to those in the calcium transient: the initial upstroke of the contraction was followed by a more gradual second component, which gave the contraction a half-dome appearance. The time to peak [Ca²⁺]_i and the time to peak contraction increased linearly with action potential duration (APD₅₀). The effects of Bay K8644 were simulated, in part, by CsCl (7.5 mM), which produced equivalent prolongation of the action potential and calcium transients. However, CsCl did not elevate diastolic [Ca²⁺]_i. To determine the mechanism of the diastolic [Ca²⁺]_i, increase, Bay K8644 was applied to aggreagates rendered quiescent by tetrodotoxin. Bay K8644 caused a graded increase in [Ca²⁺]_i, which was followed by resumption of spontaneous beating. Bay K8644 can therefore increase [Ca²⁺]_i in the absence of action potentials. We conclude that the duration of the calcium transient and its companion contraction are tightly coupled to the duration of the action potential in chick embryonic myocardial cells. Besides increasing action potential duration, Bay K8644 has the further effect of elevating diastolic [Ca²⁺]_i, which appears to contribute to the positive inotropic effect.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Intracellular calcium variations evoked by mechanical stimulation of mammalian isolated vestibular type I hair cells

The variations of intracellular free calcium concentration ([Ca²⁺]_i) were recorded on-line from guinea-pig isolated vestibular sensory cells using a fura-2 fast fluorescent photometry system, during mechanical displacements of the hair bundle. Repetitive displacements of the hair bundle towards the kinocilium (positive stimulation 7°, 300 ms, 2Hz for 10 s), revealed [Ca²⁺]_i variations detectable only in the cuticular plate. [Ca²⁺]_i increased from 105 to 145 nM. Single mechanical displacements of the hair bundle (7°, 200 ms, 0.5Hz) evoked increases of [Ca²⁺]_ifrom 50±23 nM to 139±79 (n=12). In the opposite direction, the mechanical stimulations (8°, 400ms, 0.5Hz) evoked a decrease of [Ca²⁺]_i from 68±17 nM to 37±12 nM (n= 8). The variations of [Ca²⁺]_i detected in the cuticular plate during positive displacements of the hair bundle were reversibly abolished in the presence of 100 M gentamicin and they could not be evoked in 0.1 mM calcium in the external medium. From these experiments, it has been concluded that the [Ca²⁺]_i variations recorded in the cuticular plate were due to a limited entry of calcium ions through transduction channels localized in the hair bundle. The typical kinetics of variations of [Ca²⁺]_i evoked during positive displacements of the hair bundle should account for the presence of strong calcium regulation systems in the hair bundle and cuticular plate.     

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.     

Ca2+ release in cultured rat epididymal cells during hypoosmotic swelling

Microfluorimetric studies were carried out to investigate the effects of hypoosmotic swelling on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single rat epididymal cells. In Ca²⁺-free solution containing 50 mol/l ethylenebis(oxonitrilo)tetraacetate (EGTA) hypoosmotic swelling (–160 mosmol/l) induced a transient rise in [Ca²⁺]_i which was either monophasic, biphasic or oscillatory. The [Ca²⁺]_i responses to repeated hypoosmotic stimulations followed a decremental pattern. However, if 2.5 mmol/l Ca²⁺ was admitted during the recovery period between successive stimulations, the second and the third [Ca²⁺]_i responses were slightly greater than the first. Increasing the change in osmolarity from –14±1.0 to –154±1.5 mosmol/l increased the rise in [Ca²⁺]_i but reduced the [Ca²⁺]_i response to subsequent ionomycin stimulation (4 mol/l). The swelling- and the ionomycin-induced rises in [Ca²⁺]_i followed a reciprocal pattern. It was suggested that intracellular Ca²⁺ release in response to cell swelling in the epididymal epithelium might play a role in cell volume regulation and the control of epididymal fluid osmolarity.     

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.     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonie epithelial cells (HT-29/B6)

We investigated the inhibitory action of K⁺ channel blockers on carbachol-stimulated Ca²⁺ entry into human Cl^–-secretory colonic epithelial cells (HT-29/B6). Digital imaging of the fluorescent calcium indicator dye fura-2 was performed to monitor effects of K⁺ channel blockers on cytosolic calcium in resting and carbachol-stimulated HT-29/B6 cells. Stimulation with the muscarinic agonist carbachol (100 M) caused a clearly biphasic intracellular calcium (Ca_i response: Ca_i was stimulated from resting levels (85±3 nM, n=100) to a sudden transient peak (821±44 nM) followed by a sustained plateau (317±12 nM). The maintained elevation was dependent on external Ca²⁺ and represented a new steady state between Ca²⁺ entry and exit across the plasma membrane. A monophasic Ca²⁺ response was induced in the absence of external Ca²⁺ and after the initial peak Ca_i returned to baseline. The Ca_i plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 M) or the inorganic Ca²⁺ channel blocker lanthanum (effective concentration for 50% inhibition of Ca₁ plateau EC₅₀=68±18 nM), but it was unaffected by the organic Ca²⁺ channel blockers verapamil and nifedipine. Barium, lidocaine and 4-nitro-2-(3-phenylpropylamino)benzoate (NPPB), well-known blockers of basolateral K⁺ channels of HT-29/B6 cells, rapidly and reversibly reduced carbachol-stimulated Ca²⁺ entry. The Ca_i plateau was calculated to be 50% inhibited by barium (96±2 M), lidocaine (74±3 M) and NPPB (27±10 M). The Ca_i plateau was transiently increased by 1 M and 10 M NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl^– channels (so that the membrane potential approached V _K). The resting Ca_i was transiently increased by 50 M and 300 M NPPB to 308±13 nM and 447±153 nM, respectively, suggesting that NPPB induced a Ca²⁺ release from internal Ca stores. We conclude that carbachol-stimulated Ca²⁺ entry into HT-29/B6 cells (a) requires muscarinic receptor occupation, (b) is highly sensitive to lanthanum and (c) is dependent on membrane potential and therefore inhibited by channel blockers that depolarize the cell potential. Also, the sensitivity of Ca_i levels to K⁺ channel blockers indicates that there are feedback relationships among rates of Ca²⁺ entry, activity of Ca²⁺-activated K⁺ and Cl^– channels and membrane potential.     

Pacing-Induced Non-Uniform Ca2+ Dynamics in Rat Atria Revealed by Rapid-Scanning Confocal Microscopy

Intracellular Ca²⁺ ([Ca²⁺]_i) dynamics in isolated myocytes differ between the atria and ventricles due to the distinct t-tubular distributions. Although cellular aspects of ventricular [Ca²⁺]_i dynamics in the heart have been extensively studied, little is known about those of atrial myocytes in situ. Here we visualized precise [Ca²⁺]_i dynamics of atrial myocytes in Langendorff-perfused rat hearts by rapid-scanning confocal microscopy. Of 16 fluo-4-loaded hearts imaged during pacing up to 4-Hz, five hearts showed spatially uniform Ca²⁺ transients on systole among individual cells, whereas no discernible [Ca²⁺]_i elevation developed during diastole. In contrast, the remaining hearts showed non-uniform [Ca²⁺]_i dynamics within and among the cells especially under high-frequency (4 Hz) excitation, where subcellular cluster-like [Ca²⁺]_i rises or wave-like [Ca²⁺]_i propagation occurred on excitation. Such [Ca²⁺]_i inhomogeneity was more pronounced at high-frequency pacing, showing beat-to-beat Ca²⁺ transient alternans. Despite such non-uniform dynamics, cessation of burst pacing of the atria was not followed by emergence of spontaneous Ca²⁺ waves, indicating minor Ca²⁺-releasing potentials of the sarcoplasmic reticulum (SR). In summary, rat atria display a propensity to show non-uniform [Ca²⁺]_i dynamics on systole due to impaired Ca²⁺-release from the SR and paucity of t-tubules. Our results provide an important basis for understanding atrial pathophysiology.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.