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• W1973309670 abstract "It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca2+]i) are used to control cellular processes such as gene transcription, cell proliferation, differentiation, and secretion. We recently showed that the Ca2+ oscillations in the neuroendocrine melanotrope cells of Xenopus laevis are built up by a number of discrete Ca2+ rises, the Ca2+ steps. The origin of the Ca2+ steps and their role in the generation of long-lasting Ca2+ patterns were unclear. By simultaneous, noninvasive measuring of melanotrope plasma membrane electrical activity and the [Ca2+]i, we show that numbers, amplitude, and frequency of Ca2+ steps are variable among individual oscillations and are determined by the firing pattern and shape of the action currents. The general Na+ channel blocker tetrodotoxin had no effect on either action currents or the [Ca2+]i. Under Na+-free conditions, a depolarizing pulse of 20 mm K+ induced repetitive action currents and stepwise increases in the [Ca2+]i. The Ca2+ channel blocker CoCl2 eliminated action currents and stepwise increases in the [Ca2+]i in both the absence and presence of high K+. We furthermore demonstrate that the speed of Ca2+ removal from the cytoplasm depends on the [Ca2+]i, also between Ca2+ steps during the rising phase of an oscillation. It is concluded that Ca2+ channels, and not Na+ channels, are essential for the generation of specific step patterns and, furthermore, that the frequency and shape of Ca2+ action currents in combination with the Ca2+ removal rate determine the oscillatory pattern. It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca2+]i) are used to control cellular processes such as gene transcription, cell proliferation, differentiation, and secretion. We recently showed that the Ca2+ oscillations in the neuroendocrine melanotrope cells of Xenopus laevis are built up by a number of discrete Ca2+ rises, the Ca2+ steps. The origin of the Ca2+ steps and their role in the generation of long-lasting Ca2+ patterns were unclear. By simultaneous, noninvasive measuring of melanotrope plasma membrane electrical activity and the [Ca2+]i, we show that numbers, amplitude, and frequency of Ca2+ steps are variable among individual oscillations and are determined by the firing pattern and shape of the action currents. The general Na+ channel blocker tetrodotoxin had no effect on either action currents or the [Ca2+]i. Under Na+-free conditions, a depolarizing pulse of 20 mm K+ induced repetitive action currents and stepwise increases in the [Ca2+]i. The Ca2+ channel blocker CoCl2 eliminated action currents and stepwise increases in the [Ca2+]i in both the absence and presence of high K+. We furthermore demonstrate that the speed of Ca2+ removal from the cytoplasm depends on the [Ca2+]i, also between Ca2+ steps during the rising phase of an oscillation. It is concluded that Ca2+ channels, and not Na+ channels, are essential for the generation of specific step patterns and, furthermore, that the frequency and shape of Ca2+ action currents in combination with the Ca2+ removal rate determine the oscillatory pattern. relative amplitude tetrodotoxin. Various cellular processes like gene expression, proliferation, contraction, and secretion are regulated by extracellular first-messenger molecules such as hormones, neurotransmitters, and growth factors. Regulation of these processes is often mediated by intracellular second messengers such as cAMP, inositol 1,4,5-trisphosphate, and Ca2+, which convert the extracellular signal into a cellular or subcellular response. Among second messenger-mediated signaling processes, Ca2+signaling is receiving much attention (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6134) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2250) Google Scholar, 3Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1231) Google Scholar, 4Thomas P. Surprenant A. Almers W. Neuron. 1990; 5: 723-733Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 5Tse F.W. Tse A. Hille B. Horstmann H. Almers W. Neuron. 1997; 18: 121-132Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 6Gu X. Spitzer N.C. Nature. 1995; 375: 784-787Crossref PubMed Scopus (466) Google Scholar). This signaling appears to be based on the induction of temporary and/or spatial changes in the intracellular Ca2+ concentration. These changes may be either local (i.e. sparks, blips, puffs) (7Berridge M.J. J. Physiol. (Lond.). 1997; 499: 291-306Crossref Scopus (913) Google Scholar, 8Cheng H. Lederer W.J. Cannell M.B. 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(Lond.). 1997; 499: 307-314Crossref Scopus (234) Google Scholar) or global, occurring throughout the cell, as in the case of peak-plateau phases and calcium oscillations (2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2250) Google Scholar, 7Berridge M.J. J. Physiol. (Lond.). 1997; 499: 291-306Crossref Scopus (913) Google Scholar, 17Cheek T.R. Curr. Opin. Cell Biol. 1991; 3: 199-205Crossref PubMed Scopus (30) Google Scholar, 18Berridge M.J. Dupont G. Curr. Opin. Cell Biol. 1994; 6: 267-274Crossref PubMed Scopus (198) Google Scholar, 19Bootman M.D. Berridge M.J. Lipp P. Cell. 1997; 91: 367-374Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). It is proposed that the temporal and spatial aspects of the Ca2+ signal determine (encode) which (sub)cellular process will be regulated (20Berridge M.J. Nature. 1997; 386: 759-760Crossref PubMed Scopus (387) Google Scholar). It seems that not only frequency modulation but also amplitude modulation can encode cellular effects (20Berridge M.J. Nature. 1997; 386: 759-760Crossref PubMed Scopus (387) Google Scholar, 21Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1542) Google Scholar). According to this principle, first messengers can regulate specific cellular activities by inducing distinct types of Ca2+signals. Consequently, the mechanism(s) cells use to generate different types of Ca2+ signals are of special interest. The present study concerns the relationship between plasma membrane electrical activity and Ca2+ signaling in an excitable secretory cell, the neuroendocrine melanotrope cell of Xenopus laevis. This pituitary intermediate lobe cell releases α-melanophore-stimulating hormone, a peptide that causes skin darkening in animals adapted to a black background (22Jenks B.G. Leenders H.J. Martens G.J.M. Roubos E.W. Zool. Sci. 1993; 10: 1-11PubMed Google Scholar). The cell displays intracellular Ca2+ oscillations that are regulated by neurotransmitters and neuropeptides involved in the regulation of α-melanophore-stimulating hormone release (23Roubos E.W. Comp. Biochem. Physiol. 1997; 118: 533-550Crossref Scopus (77) Google Scholar, 24Shibuya I. Douglas W.W. Endocrinology. 1993; 132: 2166-2175Crossref PubMed Scopus (42) Google Scholar, 25Shibuya I. Douglas W.W. Endocrinology. 1993; 132: 2176-2183Crossref PubMed Scopus (24) Google Scholar, 26Scheenen W.J.J.M. Jenks B.G. Roubos E.W. Willems P.H.G.M. Cell Calcium. 1994; 15: 36-44Crossref PubMed Scopus (35) Google Scholar, 27Scheenen W.J.J.M. Jenks B.G. Willems P.H.G.M. Roubos E.W. Eur. J. Physiol. 1994; 427: 244-251Crossref PubMed Scopus (43) Google Scholar). This observation has led to the conclusion that the oscillations are the driving force for secretion in this cell (24Shibuya I. Douglas W.W. Endocrinology. 1993; 132: 2166-2175Crossref PubMed Scopus (42) Google Scholar, 27Scheenen W.J.J.M. Jenks B.G. Willems P.H.G.M. Roubos E.W. Eur. J. Physiol. 1994; 427: 244-251Crossref PubMed Scopus (43) Google Scholar). In addition, Ca2+signaling is assumed to be involved in neurotransmitter-controlled biosynthesis of proopiomelanocortin, the precursor of α-melanophore-stimulating hormone (23Roubos E.W. Comp. Biochem. Physiol. 1997; 118: 533-550Crossref Scopus (77) Google Scholar, 28Dotman C.H. Cruijsen P.M.J.M. Jenks B.G. Roubos E.W. Endocrinology. 1996; 137: 4551-4557Crossref PubMed Scopus (25) Google Scholar, 29Dotman C.H. Maia A. Cruijsen P.M.J.M. Jenks B.G. Roubos E.W. Neuroendocrinology. 1997; 66: 106-113Crossref PubMed Scopus (15) Google Scholar, 30Scheenen W.J.J.M. Jenks B.G. van Dinter R.J.A.M. Roubos E.W. Cell Calcium. 1996; 19: 219-227Crossref PubMed Scopus (38) Google Scholar). The Ca2+oscillations depend on the activity of ω-conotoxin GVIA-sensitive Ca2+ channels in the plasma membrane (25Shibuya I. Douglas W.W. Endocrinology. 1993; 132: 2176-2183Crossref PubMed Scopus (24) Google Scholar, 26Scheenen W.J.J.M. Jenks B.G. Roubos E.W. Willems P.H.G.M. Cell Calcium. 1994; 15: 36-44Crossref PubMed Scopus (35) Google Scholar). Spatio-temporal studies using confocal laser-scanning microscopy have shown that each oscillation starts at the plasma membrane and is subsequently propagated as a wave to the nucleus (30Scheenen W.J.J.M. Jenks B.G. van Dinter R.J.A.M. Roubos E.W. Cell Calcium. 1996; 19: 219-227Crossref PubMed Scopus (38) Google Scholar, 31Koopman W.J.H. Roubos E.W. Jenks B.G. Cell Calcium. 1997; 22: 167-178Crossref PubMed Scopus (18) Google Scholar). The high temporal resolution of the line-scanning mode of the confocal laser-scanning microscopy has revealed that the rise phase of each oscillation is built up by a number of discrete increases referred to as Ca2+ steps (30Scheenen W.J.J.M. Jenks B.G. van Dinter R.J.A.M. Roubos E.W. Cell Calcium. 1996; 19: 219-227Crossref PubMed Scopus (38) Google Scholar, 31Koopman W.J.H. Roubos E.W. Jenks B.G. Cell Calcium. 1997; 22: 167-178Crossref PubMed Scopus (18) Google Scholar). It has been suggested that the steps are building blocks for Ca2+ signaling in theXenopus melanotrope cell (31Koopman W.J.H. Roubos E.W. Jenks B.G. Cell Calcium. 1997; 22: 167-178Crossref PubMed Scopus (18) Google Scholar). So far, no detailed information is available on how the steps contribute to the generation of distinct Ca2+ patterns. Xenopus melanotrope cells have also been shown to display bursting electrical activity (32Scheenen W.J.J.M. Jenks B.G. de Koning H.P. Vaudry H. Roubos E.W. J. Neuroendocrinology. 1994; 6: 457-464Crossref PubMed Scopus (27) Google Scholar,33Valentijn J.A. Valentijn K. Cell Calcium. 1997; 21: 241-251Crossref PubMed Scopus (12) Google Scholar). This raises the possibility that the action potentials are the driving force for local Ca2+ influxes that give rise to the stepwise build up of Ca2+ to form distinct Ca2+patterns. To test this hypothesis we have performed simultaneous measurements of electrical plasma membrane activity (cell-attached patch clamping) and Ca2+ signaling (microfluorometry). We show that the membrane action currents are Ca2+ currents, that each Ca2+ step is created by a single action current, and that the bursting pattern of Ca2+ currents, in combination with the Ca2+removal rate, determines the shape of each oscillation. Young-adult (8 months of age) male and female specimens of X. laevis, raised in our department under standard laboratory conditions, were adapted to a dark background for at least three weeks before the experiments, under continuous illumination, at 22 °C. The animals were fed weekly with beef heart. All experiments have been carried out under the guidelines of Dutch laws concerning animal welfare. Animals were anesthetized in a solution containing 0.1% (w/v) MS222 (3-aminobenzoic acid ethyl ester; Sigma). To remove blood cells, the animals were perfused withXenopus Ringer's solution containing 112 mmNaCl, 2 mm KCl, 2 mm CaCl2, 15 mm Ultral-HEPES (Calbiochem), 10 mm glucose, and 0.025% (w/v) MS222 (pH 7.4). After decapitation, neurointermediate lobes of the pituitary gland were rapidly dissected and rinsed four times in XL L15 culture medium consisting of 76% (v/v) L15 medium (Life Technologies, Inc.), 1% (v/v) kanamycin solution (Life Technologies, Inc.), 1% (v/v) antibiotic/antimyotic solution (Life Technologies, Inc.), 2 mm CaCl2, and 10 mm glucose (pH 7.4). After an incubation period of 45 min in Xenopus Ringer's solution without CaCl2 and with 0.25% (w/v) trypsin (Life Technologies, Inc.), the lobes were dissociated by gentle trituration with a siliconized Pasteur's pipette. Then, the resulting cell suspension was filtered, followed by centrifugation for 10 min at 500 rpm. The pellet was resuspended in XL L15 culture medium (80 μl/lobe-equivalent), and the cells were plated on coverslips coated with poly(l-lysine) (Sigma;M r > 300 kDa) in aliquots equivalent to 1 lobe/coverslip, yielding approximately 10,000 cells/coverslip. The cells were allowed to settle for 1–1.5 h in an incubator, at 22 °C. Then 2 ml of XL L15 medium containing 10% fetal calf serum was added to each coverslip, and the cells were incubated for another 3 days at 22 °C before use. A microscopic setup for combined, time-coordinated microfluorometric and electrophysiological experiments was used to simultaneously measure changes in the [Ca2+]i and action currents (current waveforms that represent action potentials). To measure changes in the [Ca2+]i, cells were loaded with 4 μm fura-2/AM (Molecular Probes, Leiden, NL) in Xenopus Ringer's solution containing 1 μmPluronic F127 (Molecular Probes) (34Poenie M. Alderton J. Steinhardt R. Tsien R. Science. 1986; 233: 886-889Crossref PubMed Scopus (369) Google Scholar) for 30 min at 22 °C. After loading, cells were washed with Xenopus Ringer's solution to remove nonhydrolyzed fura-2/AM. Thereafter, cells were placed under continuous superfusion with Xenopus Ringer's solution (1 ml/min) on the stage of an upright microscope (Zeiss Axioskop FS, Göttingen, Germany). Unattached cells were removed, and attached cells were allowed to equilibrate for 30 min before the start of an experiment. During an experiment, cells were alternately exposed to excitation light from a multiwavelength illumination system (T.I.L.L. photonics, polychrome II, Planegg, Germany) at wavelengths of 355 and 380 nm. The fluorescence emission at 510 nm was measured with a photomultiplier tube (Hamamatsu Photonics K.K., R928, Japan) at a sampling rate of 50 Hz. The ratio of the emission intensities (355 nm/380 nm) was used as a measure for changes in the [Ca2+]i. Electrophysiological recordings were performed using the cell-attached recording configuration of the patch-clamp technique (35Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15065) Google Scholar). In this cell-attached configuration, biphasic wave forms, the action currents reflecting action potentials, were recorded without disturbing the intracellular environment. All recordings were made with a pipette potential of 0 mV using an EPC-9 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany). Data were filtered with the built-in 4-pole Bessel filter of the EPC-9 at 3 kHz. Synchronized acquisition of both microfluorometric and electrophysiological data was performed with an Apple Macintosh PowerPC 8200/120 with Pulse/Pulsefit software (version 8.07; HEKA). Patch electrodes with a resistance of 4–6 megaohms were pulled from borosilicate glass capillaries (GC150–15; Clark Electromedical Instruments, Pangbourne, UK) using a Narishige PP-83 pipette puller (Narishige Scientific Instrument Laboratories, Tokyo, Japan). They were filled with Xenopus Ringer's solution. To study the calcium dynamics during loading, cells (n= 4) were loaded with fura-2 via a patch pipette in the whole-cell voltage-clamp configuration as described by Helmchen et al.(36Helmchen F.J. Borst G.G. Sakmann B. Biophys. J. 1997; 72: 1458-1471Abstract Full Text PDF PubMed Scopus (232) Google Scholar). The pipette solution contained 100 μmfura-2, 112 mm CsCl, 1.8 mm MgCl2, 0.2 mm MgATP, 10 mm Ultral-HEPES (pH 7.4, adjusted with CsOH). The extracellular solution consisted of 93 mm tetraethylammonium chloride, 2 mmCaCl2, 5 mm CsCl, 15 mmUltral-HEPES, 2 mm MgCl2, 10 mmglucose (pH 7.4, adjusted with tetraethylammonium hydroxide). Background fluorescence intensity was determined when the pipette was still in the cell-attached configuration. After break-in, Ca2+ influx was triggered every 15 s by a depolarizing pulse from −80 to 0 mV with a duration of 100 ms. At the same time fura-2 fluorescence emission intensities were measured at 355 and 380 nm excitation as described above. The ratio of the emission intensities (355 nm/380 nm) was used to determine the decay time constants (τ) and the relative amplitude (A r)1 of the evoked Ca2+ transients at different fura-2 concentrations. Drug-containing solutions were applied by local perfusion from a wide-mouthed glass pipette (inner diameter 0.8 mm) placed about 100 μm from the recorded cell. The level of the bath solution was kept constant by means of a suction device. In Na+-free conditions, NaCl was replaced by an equiosmotic amount of N-methylglucamine. To block Na+channels, 1 μm tetrodotoxin (TTX) was used, whereas Ca2+ channels were blocked with 2 mmCoCl2. To keep the osmolarity constant, the concentration of NaCl was adjusted when high K+ concentrations (20 mm) were used. All chemicals were from Sigma, unless stated otherwise. Because it is known that the concentration of exogeneous Ca2+ buffers like fura-2 can alter the amplitude and kinetics of Ca2+ transients (36Helmchen F.J. Borst G.G. Sakmann B. Biophys. J. 1997; 72: 1458-1471Abstract Full Text PDF PubMed Scopus (232) Google Scholar, 37Sabatini B.L. Regehr W.G. Biophys. J. 1998; 74: 1549-1563Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) we checked whether this also holds for Xenopusmelanotropes. Cells (n = 4) were loaded with fura-2 via patch pipettes in the whole-cell voltage-clamp configuration. After break-in, Ca2+ influx was triggered every 15 s by depolarizing pulses from −80 to 0 mV with a duration of 100 ms (Fig. 1 A, circles). The Ca2+-insensitive fluorescence (F 355) was used to monitor the diffusion of fura-2 into the cell (Fig. 1 A). We assumed that the concentration of fura-2 in the cell was equal to the fura-2 pipette concentration whenF 355 reached a plateau. AsF 355 followed an exponential time course, the fura-2 concentration during loading could be calculated at any given time. The ratio of the emission intensities (355 nm/380 nm;eg. Fig. 1 A, inset) was used to determine the decay time constants (Fig. 1 B, τ (tau)) and relative amplitude (Ar =R top/R basal; Fig. 1 C) of the evoked transients at different fura-2 concentrations. The inset in Fig. 1 A shows a transient directly measured after break-in (Fig. 1 A;black circle). To determine τ, single exponentials were fitted to the decays of the Ca2+ transients with a fit range that started within 20 ms after the peak and extended to 5 s after the peak. The dependence of τ on the [fura-2] was well described by a linear relationship according to τ = A +B × [fura-2] (Fig. 1 B;line) with A = 2.5 ± 0.37,B = 4.5 ×10−5 ± 8 × 10−3, and a linear correlation coefficient (Pearson'sr; r p) (38Press W.H. Flannery B.P. Teukolsky S.A. Vetterling W.T. Numerical Recipes in Pascal: The Art of Scientific Computing. Cambridge University Press, Cambridge, UK1992Google Scholar) of 0.0024. This almost horizontal line indicates that τ was not dependent on the [fura-2]. The relationship between Ar and the [fura-2] was described by the line Ar = C + D × [fura-2] (Fig. 1 C) with C = 1.38 ± 0.027, D = −0.002 ± 0.0006, and r p = −0.67. This means that there was only a small decrease (D = −0.002 ± 0.0006) in amplitude during loading. However, no relationship between Ar and τ was found (Fig. 1 D; horizontal line, τ =E + F × Ar withE = 2.5 ± 2.93, F = 0.0019 ± 2.27, and r p = 0.0024). About 80% of the single melanotrope cells derived from the pituitary gland of X. laevis appear to display spontaneous Ca2+ oscillations (26Scheenen W.J.J.M. Jenks B.G. Roubos E.W. Willems P.H.G.M. Cell Calcium. 1994; 15: 36-44Crossref PubMed Scopus (35) Google Scholar). In the present study, a total of 42 oscillating cells were studied to investigate the detailed nature and origin of the Ca2+ patterns in individual cells loaded with the Ca2+ indicator fura-2/AM. When recording the [Ca2+]i at a sampling rate of 6 s, smooth oscillations with a fixed frequency and amplitude were observed (e.g. Fig. 2 A). With a much higher temporal resolution of 20 ms, cells showed highly complex Ca2+ oscillation patterns with strong inter- and intracellular differences (Fig. 2, B–D). In most cells (37 of 42), oscillations did not appear smooth but showed stepwise increases, the Ca2+ steps (Fig. 2, B and C). In only a few cases (5 of 42), the oscillations reached the peak amplitude after one discrete rise in the [Ca2+]i (Fig. 2 D). Oscillations of different cells not only varied in frequency and relative amplitude but also in the number of steps building up an oscillation, which ranged from 1 (Fig. 2 D) to 17 (Fig. 8 A). Within a given cell, the number of Ca2+ steps building up a Ca2+ oscillation can also vary (Fig. 2 E). Fig. 2 E shows that the amplitude of the oscillatory pattern may not necessarily be determined by the number of steps building up an oscillation. For example, the first and second oscillation shown in Fig. 2 E have the same relative amplitude (Ar= 1.39), whereas in the first oscillation two more steps are required to reach this amplitude. The amplitude of the oscillation displayed in Fig. 2 D is even bigger (Ar = 1.69) than that shown in Fig. 2 E, whereas only one discrete rise in the [Ca2+]i can be observed. Moreover, steps are not only present during the rising phase of an oscillation but also on top of an oscillation (Fig. 2, B and C). The number of steps on top of an oscillation is variable (e.g. Fig. 2 B; first versus second oscillation), and this parameter determines the duration of an oscillation within a given cell. In addition, the average step interval between cells can vary. For example, the step interval calculated from the first 15 steps in Fig. 2 B is 1.88 ± 0.24 s (mean ±S.E.). This is significantly higher (Student's t test; p< 0.0001) than the step interval calculated from the first 15 steps in Fig. 2 C, which is 0.56 ± 0.09 s (mean ± S.E.).Figure 8Kinetics of Ca2+ removal from the cytoplasm changes during a Ca2+ oscillation. The velocity of Ca2+ removal from the cytoplasm (Vd) was analyzed both during the rising phase and the declining phase of oscillations. A, three oscillations (I, II, and III) containing 27 steps (seenumbers) were used to study Vd. Dotted lines represent the linear fits of the interval [R t,n, R b,n+1] (seepanel B). Step marked by an asterisk (*) was omitted from analysis (bad fit). The box indicates the part that is enlarged to form panel B. B, parameters used to describe the step kinetics. R b,1, the resting fura-2 emission ratio; R t,n, the fura-2 emission ratio at the top of step n;R b,n, the fura-2 emission ratio just before occurrence of step n; and dx n, the time needed for the fura-2 emission ratio to drop fromR t,n to R b,n+1. Anasterisk (*) marks the time needed for the fura-2 emission ratio to rise from R b,n to R t,n.C, V d,linear, the velocity of Ca2+ removal after a Ca2+ step determined by a linear fit (for details, see “Results”), plotted against the step number. D, a linear correlation was found betweenV d,linear and R t,n, the fura-2 emission ratio at the top of step n.E, a clear linear correlation was found betweenV d,linear and V d,exp. Vd,exp is the velocity of Ca2+ removal during the declining phase of oscillations determined using a first order exponential function (see Eq. 1 under “Results”).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test whether fura-2 loading influences the electrical membrane activity, the firing pattern was checked in unloaded melanotropes. Using the cell-attached patch configuration, clear bursts of spontaneous “action currents,” representing action potentials, were observed in unloaded, single melanotropes (Fig. 2 F;n = 6). These bursts were similar to the bursts observed during combined measurements with fura-2-loaded cells (see Fig. 3 A), indicating that loading the cells with fura-2 does not alter the firing behavior. To study the relation between the electrical activity and Ca2+ oscillations of the same cell, electrophysiological measurements were combined with simultaneous measurements of changes in the [Ca2+]i (Fig. 3 A;n = 42). To check for possible pipette-induced changes in the original Ca2+ signal, each combined experiment was preceded by a Ca2+ measurement alone. Fig. 3 Ashows that the bursts of electrical activity are directly related to the Ca2+ oscillations. From looking in detail (Fig. 3 B), it is evident that each action current is accompanied by a discrete rise in the [Ca2+]i. After a burst of action currents, the [Ca2+]i smoothly returns to the basal level. During some measurements, action current firing changed from a bursting mode into continuous firing (Fig. 4 A), resulting in the disappearance of Ca2+ oscillations and a steady high [Ca2+]i. Because this phenomenon was never observed during the Ca2+ measurements preceding the combined measurements, we conclude that these particular changes in the firing pattern could have been induced by the pipette. Therefore, such recordings were discarded. Nonetheless, it is interesting to note that even at this high Ca2+ level, the tight relationship between action current firing and the occurrence of Ca2+steps was maintained (Fig. 4 B). To determine the nature of the inward currents, the Na+ channel blocker TTX and the inorganic Ca2+channel blocker Co2+ were added. 1 μm TTX did not have an effect on either action currents or Ca2+oscillations in any of the cells measured (Fig. 5 A; n = 11). On the other hand, applying 2 mm CoCl2 clearly abolished both action currents and Ca2+ oscillations in every cell studied (Fig. 5 B; n = 15). In the complete absence of extracellular Na+ (Na+replaced by N-methyl-d-glucamine), no action currents or Ca2+ oscillations were observed (Fig. 6 A; n = 4). Under this Na+-free condition, action currents and a rise in the [Ca2+]i could still be induced by a depolarizing K+ (20 mm) pulse (Fig. 6 A; n = 4). The rise in the [Ca2+]i was clearly built up by a number of Ca2+ steps, each accompanied by an action current (Fig. 6 B).Figure 6Effect of extracellular Na+removal, alone or in combination with a depolarizing K+pulse, on action current firing and the [Ca2+]i. Combined measurements of action currents (upper trace) and [Ca2+]ichanges (lower trace) were obtained as in Fig. 3. Na+-free medium (Na+ replaced byN-methyl-d-glucamine (NMDG)) was applied to the cells as indicated by the upper horizontal bar in panel A. To depolarize the cell under this Na+-free condition, a 20 mm K+pulse was given, as indicate by the lower horizontal bar in panel A. A, extracellular Na+ removal blocked both action currents (upper trace) and Ca2+ oscillations (lower trace). Under this Na+-free condition, a depolarizing K+ pulse induced action currents and a rise in the [Ca2+]i. B, region indicated in detail showing the action currents and the Ca2+ steps building up the increase in the [Ca2+]i during the high K+ treatment under the Na+-free condition.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because action current measurements were performed simultaneously with Ca2+ measurements, a link between the shape of an action current and the amplitude of a Ca2+ step could be demonstrated. The upper traceof Fig. 7 A shows the action currents reflecting the Ca2+ steps shown in the lower trace of Fig. 7 A. Whereas the amplitude of the successive action currents decreased, the amplitude of the accompanying Ca2+ steps increased. The relative difference in the ratio values between the start and the top of a Ca2+ step (Ar =R top/R basal); lower trace of Fig. 7 A) was taken as the step amplitude. To determine the amount of charge entering the cell during an action current, the peak areas of the downward action currents were integrated. In Fig. 7 B the peak area of each action current was plotted against the relative amplitude of the Ca2+steps in Fig. 7 A. The peak areas of the three successive action currents increased, and the amplitude of the Ca2+steps increased. A clear linear relation was found between the current peak area and the Ca2+ step size. The calcium removal kinetics have been studied to investigate the role of this removal in shaping the calcium oscillations. The presence of a discontinuous (i.e. stepping) Ca2+ oscillation presents a unique opportunity to analyze the speed of removal of Ca2+ from the cytoplasm" @default.
• W1973309670 created "2016-06-24" @default.
• W1973309670 creator A5028790221 @default.
• W1973309670 creator A5035876859 @default.
• W1973309670 creator A5056236361 @default.
• W1973309670 creator A5073623207 @default.
• W1973309670 creator A5088296029 @default.
• W1973309670 creator A5091914657 @default.
• W1973309670 date "1998-10-01" @default.
• W1973309670 modified "2023-10-15" @default.
• W1973309670 title "Action Currents Generate Stepwise Intracellular Ca2+Patterns in a Neuroendocrine Cell" @default.
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