INTRODUCTION

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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 (1-6). 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) (7-16) or global, occurring throughout the cell, as in the case of peak-plateau phases and calcium oscillations (2, 7, 17-19).

It is proposed that the temporal and spatial aspects of the Ca²⁺ signal determine (encode) which (sub)cellular process will be regulated (20). It seems that not only frequency modulation but also amplitude modulation can encode cellular effects (20, 21). 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 -melanophore-stimulating hormone, a peptide that causes skin darkening in animals adapted to a black background (22). The cell displays intracellular Ca²⁺ oscillations that are regulated by neurotransmitters and neuropeptides involved in the regulation of -melanophore-stimulating hormone release (23-27). This observation has led to the conclusion that the oscillations are the driving force for secretion in this cell (24, 27). In addition, Ca²⁺ signaling is assumed to be involved in neurotransmitter-controlled biosynthesis of proopiomelanocortin, the precursor of -melanophore-stimulating hormone (23, 28-30). The Ca²⁺ oscillations depend on the activity of -conotoxin GVIA-sensitive Ca²⁺ channels in the plasma membrane (25, 26). 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 (30, 31). 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 (30, 31). It has been suggested that the steps are building blocks for Ca²⁺ signaling in the Xenopus melanotrope cell (31). 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 (32, 33). 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.

	EXPERIMENTAL PROCEDURES

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Animals-- 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.

Cell Culture-- 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 with Xenopus Ringer's solution containing 112 mM NaCl, 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.

Measurements of Action Currents and Changes in the [Ca²⁺]_i-- 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).

Chemicals-- 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 mM CoCl₂. 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.

	RESULTS

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Calcium Dynamics and Fura-2-loading-- Because it is known that the concentration of exogeneous Ca²⁺ buffers like fura-2 can alter the amplitude and kinetics of Ca²⁺ transients (36, 37) we checked whether this also holds for Xenopus melanotropes. 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. 1A, circles). The Ca²⁺-insensitive fluorescence (F₃₅₅) was used to monitor the diffusion of fura-2 into the cell (Fig. 1A). We assumed that the concentration of fura-2 in the cell was equal to the fura-2 pipette concentration when F₃₅₅ reached a plateau. As F₃₅₅ 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. 1A, inset) was used to determine the decay time constants (Fig. 1B,  (tau)) and relative amplitude (Ar = R_top/R_basal; Fig. 1C) of the evoked transients at different fura-2 concentrations. The inset in Fig. 1A shows a transient directly measured after break-in (Fig. 1A; black circle). To determine , 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  on the [fura-2] was well described by a linear relationship according to  = A + B × [fura-2] (Fig. 1B; line) with A = 2.5 ± 0.37, B = 4.5 ×10⁵ ± 8 × 10³, and a linear correlation coefficient (Pearson's r; r_p) (38) of 0.0024. This almost horizontal line indicates that 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. 1C) 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  was found (Fig. 1D; horizontal line,  = E + F × Ar with E = 2.5 ± 2.93, F = 0.0019 ± 2.27, and r_p = 0.0024).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Calcium dynamics and fura-2 loading. Xenopus melanotropes were loaded with 100 µM fura-2 via patch pipettes in the whole-cell voltage-clamp configuration. A, the Ca2+-insensitive isosbestic fluorescence (F355) was used to monitor the loading of cells with 100 µM fura-2. Background fluorescence intensity was determined in the cell-attached mode (dashed line). After break-in, Ca2+ influx was triggered every 15 s by depolarizing pulses from 80 to 0 mV with a duration of 100 ms (indicated by circles). The inset shows the ratiometric Ca2+ transient (F355/F380) measured 15 s after break-in (black circle in F355 trace).  is obtained from the single exponential function describing the decay phase of the transient. The Ar in arbitrary units (AU) is given as Rtop/Rbasal. B, the decay time constants  (±S.D.) of the ratiometric Ca2+ transients as a function of the [fura-2]. The line  = A + B × [fura-2], with A = 2.5 ± 0.37, B = 4.5 × 105 ± 8 ×103, and rp = 0.0024, describes the relationship between  and the [fura-2]. C, the Ar of the ratiometric Ca2+ transients as a function of the [fura-2]. The line Ar = C + D × [fura-2], with C = 1.38 ± 0.027, D = 0.002 ± 0.0006, and rp = 0.67, describes the relationship between Ar and the [fura-2]. D, the decay time constants  (±S.D.) of the ratiometric Ca2+ transients as a function of Ar. The line  = E + F × Ar, with E = 2.5 ± 2.93, F = 0.0019 ± 2.27, and rp = 0.0024, describes the relationship between  and Ar (for details, see "Results").

Spontaneous Ca²⁺ Patterns and Electrical Activity-- About 80% of the single melanotrope cells derived from the pituitary gland of X. laevis appear to display spontaneous Ca²⁺ oscillations (26). 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. 2A). 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. 2D). 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. 2D) to 17 (Fig. 8A). Within a given cell, the number of Ca²⁺ steps building up a Ca²⁺ oscillation can also vary (Fig. 2E). Fig. 2E 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. 2E 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. 2D is even bigger (Ar = 1.69) than that shown in Fig. 2E, 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. 2B; 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. 2B 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. 2C, which is 0.56 ± 0.09 s (mean ± S.E.).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Patterns of spontaneous Ca2+ oscillations and electrical activity observed in single Xenopus melanotrope cells. Relative changes in the [Ca2+]i measured in single melanotropes loaded with the Ca2+ indicator fura-2/AM. A, oscillations measured with a sampling interval of 6 s. The oscillations look smooth and regular. B-E, complex oscillation patterns observed with a sampling interval of 20 ms. Each oscillation is built up by discrete rises in the [Ca2+]i, the Ca2+ steps. Oscillatory patterns differ because of variation in the number of steps building up an oscillation, the number of steps on top of an oscillation (e.g. trace B), and variation in the step interval (compare traces B and C). E, the relative amplitude (Ar = Rtop/Rbasal in arbitrary units) of the oscillation is not necessarily determined by the number of steps. In the first oscillation, five steps are needed, whereas in the second oscillation, only three steps are sufficient to reach the same relative amplitude. F, bursting electrical activity of an unloaded melanotrope measured using the cell-attached patch-clamp configuration.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Simultaneous measurements of spontaneous electrical activity and spontaneous changes in the [Ca2+]i. Electrophysiological recordings of action currents, representing action potentials, and recordings of changes in the [Ca2+]i obtained by combining the cell-attached configuration of the patch-clamp technique with Ca2+ microfluorometry. A, bursts of spontaneous action currents (upper trace) and the associated Ca2+ pattern (lower trace). B, detail showing action currents (upper trace) accompanied by Ca2+ steps (lower trace) building up an oscillation.

Relation between Ca²⁺ Patterns and Action Potential Firing-- 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. 3A; 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. 3A shows that the bursts of electrical activity are directly related to the Ca²⁺ oscillations. From looking in detail (Fig. 3B), 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.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Changes in the action current firing mode relate to changes in the [Ca2+]i. The figure shows a combined measurement of action currents and changes in the [Ca2+]i as described in Fig. 3. A, a change of action current firing from a bursting pattern into a continuous mode (upper trace) resulted in the disappearance of Ca2+ oscillations and a steady high Ca2+ level (lower trace). B, region indicated in detail showing that during continuous action current firing (upper trace), each action current led to a Ca2+ step at a high Ca2+ level (lower trace).

Involvement of Na⁺ and Ca²⁺ in Step Generation-- 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. 5A; n = 11). On the other hand, applying 2 mM CoCl₂ clearly abolished both action currents and Ca²⁺ oscillations in every cell studied (Fig. 5B; 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. 6A; 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. 6A; 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. 6B).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Effect of Na+ channel blocker TTX and Ca2+ channel blocker Co2+ on action current firing and the [Ca2+]i. The figure shows simultaneous measurements of action currents (upper trace) and [Ca2+]i changes (lower trace). A, 1 µM TTX was applied to the cells as indicated by the horizontal bar. TTX did not have an effect on either action current firing or on Ca2+ oscillations. B, by adding 2 mM CoCl2 in the extracellular solution, Co2+ was applied to the cells as indicated by the horizontal bar. Co2+ reversibly blocked both action current firing (upper trace) and Ca2+ oscillations (lower trace).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect 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+]i changes (lower trace) were obtained as in Fig. 3. Na+-free medium (Na+ replaced by N-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.

Relation between the Shape of an Action Current and the Amplitude of a Ca²⁺ Step-- 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 trace of Fig. 7A shows the action currents reflecting the Ca²⁺ steps shown in the lower trace of Fig. 7A. 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. 7A) 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. 7B the peak area of each action current was plotted against the relative amplitude of the Ca²⁺ steps in Fig. 7A. 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.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Relation between the shape of action currents and the amplitude of Ca2+ steps. Combined measurements of action currents (upper trace) and Ca2+ steps (lower trace) carried out as in Fig. 3. A, the peak areas of the action currents were determined by integration, and the relative amplitude (Ar in arbitrary units (AU)) of the Ca2+ steps was calculated by dividing Rtop/Rbasal. B, graph showing the positive correlation between the action current peak areas and the amplitude of the Ca2+ steps.

Kinetics of Ca²⁺ Removal during a Ca²⁺ Oscillation-- 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 (V_d) during the rising phase of a Ca²⁺ oscillation. The speed of Ca²⁺ removal was analyzed both during the rising phase following each Ca²⁺ step and during the declining phase, bringing Ca²⁺ back to basal level. Fig. 8A shows an example of a Ca²⁺ oscillation built up by steps. In total, 27 steps can be distinguished distributed over three individual oscillations (numbered I, II, and III).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Kinetics 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 (see numbers) were used to study Vd. Dotted lines represent the linear fits of the interval [Rt,n, Rb,n+1] (see panel 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. Rb,1, the resting fura-2 emission ratio; Rt,n, the fura-2 emission ratio at the top of step n; Rb,n, the fura-2 emission ratio just before occurrence of step n; and dxn, the time needed for the fura-2 emission ratio to drop from Rt,n to Rb,n+1. An asterisk (*) marks the time needed for the fura-2 emission ratio to rise from Rb,n to Rt,n. C, Vd,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 between Vd,linear and Rt,n, the fura-2 emission ratio at the top of step n. E, a clear linear correlation was found between Vd,linear and Vd,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").

(Eq. 1)

(Eq. 2)

(Eq. 3)

	DISCUSSION

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Calcium Dynamics and Fura-2 Loading-- Because it is known that the concentration of exogeneous Ca²⁺ buffers like fura-2 can alter the amplitude and kinetics of Ca²⁺ transients in presynaptic terminals of neurons (36, 37), we checked whether this holds also for the neuroendocrine melanotrope cells of Xenopus. The  values of the decays of the Ca²⁺ transients are independent of the [fura-2] and are in the range of 2-5 s. Only a very small decrease in relative amplitude (Ar) was found during loading. However, absolutely no relationship between  and Ar is present. Therefore, we conclude that the intrinsic Ca²⁺ removal in Xenopus melanotropes is rather slow and not altered by the loading with fura-2.

Ca²⁺ Patterns-- Ca²⁺ oscillations in Xenopus melanotropes have a frequency of about 1-3/min, and within each cell, have a rather stable amplitude (24-27). They have a smooth appearance when observed during Ca²⁺ measurements using a sampling interval of 6 s (see Fig. 1A). In the present study, these oscillations have been studied in detail, with particular attention to their shape and the mechanisms that generate this shape. By performing continuous measurements with a high temporal resolution (20 ms) clear inter- and intracellular variations in Ca²⁺ patterns were observed. The Ca²⁺ oscillations did not appear smooth anymore but were built up by discrete changes in the [Ca²⁺]_i, the Ca²⁺ steps. These findings extend recent confocal line scanning experiments (30, 31), which suggested that the rising phase of oscillations are built up by three to four of these discrete steps. The present study, where the microfluorometric method allows continuous Ca²⁺ measurements with a high temporal resolution, shows that the number of steps during an oscillation is variable and can be as high as 17. The oscillations had discrete rises not only during the rising phase of an oscillation but often also on top of the oscillation. This has not been reported for melanotropes or other secretory cells before.

The Role of Action Potentials in the Generation of Specific Ca²⁺ Oscillation Patterns-- To study the ability of a cell to transfer information in the form of complex [Ca²⁺]_i patterns, it is important to understand how the Ca²⁺ patterns are generated. From fast line scanning experiments, it is known that the Ca²⁺ oscillations in Xenopus melanotropes can travel through the cell as multiple waves from the plasma membrane into the nucleus (30, 31). The Ca²⁺ oscillations are inhibited by the N-type Ca²⁺ channel blocker -conotoxin GVIA (26) and not by the L-type Ca²⁺ channel blocker nifedipine (26). This indicates that they originate at the membrane by Ca²⁺ influx through N-type Ca²⁺ channels. It has been hypothesized that action potentials cause the opening of the voltage-operated N-type Ca²⁺ channels (32, 33, 39, 40). However, a direct relation between action potentials and Ca²⁺ oscillations has never been shown in pituitary melanotropes. Until now, the action potentials in Xenopus melanotropes observed have been induced by depolarizing pulses using the whole-cell patch-clamp technique (32, 33), or they have been measured using the perforated-patch configuration (33). In the present study, the cell-attached configuration of the patch-clamp technique was used to prevent any disturbances of the intracellular environment. In this patch configuration, bursts of action currents representing bursts of action potentials were observed. The bursting behavior was not induced by fura-2 loading as both loaded and unloaded cells displayed this firing behavior. The combined patch-clamp and Ca²⁺ microfluorometry showed that each action current is generating a discrete, stepwise increase in the [Ca²⁺]_i. In this way, a burst of action currents builds up a full-sized Ca²⁺ oscillation. Also the Ca²⁺ steps on top of an oscillation appeared to be generated by action currents. After each burst of action currents, the [Ca²⁺]_i smoothly returned to the basal level. In some combined experiments, the Ca²⁺ signal went up and stayed high. At the same time, action current firing changed from a bursting pattern into continuous firing, as observed previously in Xenopus melanotropes (32, 33). These changes may have been induced by the patch pipette because such sustained Ca²⁺ elevations were never observed in the control Ca²⁺ measurements preceding the combined experiments.

Currents Underlying Action Potentials-- Mathematical models containing Na⁺ channels (48, 49) predict that changes in the Na⁺ current lead to other bursting patterns. However, TTX, which blocks most of the Na⁺ currents in Xenopus melanotropes (33), had no effect on either the electrical firing or the Ca²⁺ oscillations. Blocking the Ca²⁺ channels with CoCl₂ on the other hand fully blocked both phenomena. Under Na⁺-free conditions, electrical activity and [Ca²⁺]_i changes were absent, probably because of a general hyperpolarization. Although TTX-insensitive Na⁺ currents are present in Xenopus melanotropes (33), we showed that even in the complete absence of Na⁺, a K⁺-evoked depolarization induced Ca²⁺ action currents and stepwise increases in the [Ca²⁺]_i, which looked similar to those seen under normal conditions. This observation indicates that the machinery for generating the action currents and steps is still fully functional. Although these findings suggest a role for Na⁺ only in setting the excitability of the plasma membrane, with Ca²⁺ determining the bursting behavior, we cannot rule out a contribution by TTX-insensitive Na⁺ channels.

The Role of Ca²⁺ Removal in Generating Specific Ca²⁺ Patterns-- In this study we show for the first time that during the rising phase of a Ca²⁺ oscillation, the speed of Ca²⁺ removal is not constant but depends on the [Ca²⁺]_i. With increasing [Ca²⁺]_i the removal speed linearly increases. This up-regulation plays a role in shaping the Ca²⁺ oscillation. During the rising phase of an oscillation, each Ca²⁺ influx episode leaves the [Ca²⁺]_i at a higher level than the preceding episode. At the top of an oscillation the influx and removal of Ca²⁺ become balanced because of the up-regulation of the removal speed, and consequently influx episodes no longer give a net increase in the [Ca²⁺]_i. The relatively slow removal at a low [Ca²⁺]_i leads to the stepwise increase. Interestingly, this process fits with mathematical models (48) that predict that, although the electrical bursting patterns look the same, the shapes of Ca²⁺ oscillations could be very different depending on how fast Ca²⁺ is removed from the cytoplasm. If the Ca²⁺ removal rate is low, the [Ca²⁺]_i increases, reaching a maximum near the end of a burst of action potentials. If the Ca²⁺ removal is very fast, each action potential gives rise to a single Ca²⁺ transient. In these models fixed values for the speed of Ca²⁺ removal were used. However, we now have demonstrated that the Ca²⁺ removal rate is not fixed during an oscillation.

Conclusion-- This study shows that bursting firing in combination with the calcium removal rate determine the shape of a Ca²⁺ oscillation. In this way individual Xenopus melanotrope cells can generate different Ca²⁺ oscillatory patterns. We propose that not only the frequency and amplitude of the Ca²⁺ oscillations serve to encode cellular regulatory information but also other oscillation pattern variables such as number of Ca²⁺ steps, the step amplitude, the step frequency, and the speed of Ca²⁺ removal. Therefore, the information storage capacity of the Ca²⁺ signaling seems much more complex than previously thought. Currently, studies are being carried out on the modulation of the Ca²⁺ patterns in Xenopus melanotropes by various regulatory neurotransmitters and under different conditions of adaptation of the animal to background light intensity.