Melanotrope Cells Of Xenopus Laevis Research Articles

Pituitary melanotrope cells of Xenopus laevis are of neural ridge origin and do not require induction by the infundibulum

Classical studies in amphibians have concluded that the endocrine pituitary and pars intermedia are derived from epithelial buccal epidermis and do not require the infundibulum for their induction. These studies also assumed that the pituitary is not subsequently determined by infundibular induction. Our extirpation, auto-transplantation and immunohistochemical studies with Xenopus laevis were initiated to investigate early presumptive pituitary development. These studies were conducted especially with reference to the pars intermedia melanotrope cell’s induction, and its production and release of α-melanophore stimulating hormone (α-MSH) from the precursor protein proopiomelanocortin (POMC). Auto-transplantation studies demonstrated that the pituitary POMC-producing cells are determined at a stage prior to pituitary-infundibular contact. The results of experiments involving the extirpation of the presumptive infundibulum also indicated that the infundibulum is not essential for the differentiation of POMC-producing cells. We also demonstrated that early pituitary development involves adherence to the prechiasmatic area of the diencephalon with the pituitary placode growing in a posterior direction toward the infundibulum where contact occurs at Xenopus stage 39/40. Overall, our studies provide a model for early tissue relations among presumptive pituitary, suprachiasmatic nucleus, pars tuberalis and infundibulum during neurulation and later neural tube stages of development. It is hypothesized that the overlying chiasmatic area suppresses pituitary differentiation.

ERK-Regulated Double Cortin-Like Kinase (DCLK)-Short Phosphorylation and Nuclear Translocation Stimulate POMC Gene Expression in Endocrine Melanotrope Cells

We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr kinase predominantly expressed in the brain, is downstream of the ERK signaling pathway and is involved in proopiomelanocortin gene (POMC) expression in endocrine pituitary melanotrope cells of Xenopus laevis. Melanotropes form a well-established model to study physiological aspects of neuroendocrine plasticity. The amphibian X. laevis adapts its skin color to the background light intensity by the release of α-MSH from the melanotrope cell. In frogs on a white background, melanotropes are inactive but they are activated during adaptation to a black background. Our results show that melanotrope activation is associated with an increase in DCLK-short mRNA and with phosphorylation of DCLK-short at serine at position 30 (Ser-30). Upon cell activation phosphorylated Ser-30-DCLK-short was translocated from the cytoplasm into the nucleus, and the ERK blocker U0126 inhibited this process. The mutation of Ser-30 to alanine also inhibited the translocation and reduced POMC expression, whereas overexpression stimulated POMC expression. This is the first demonstration of DCLK-short in a native endocrine cell. We conclude that DCLK-short is physiologically regulated at both the level of its gene expression and protein phosphorylation and that the kinase is effectively regulating POMC gene expression upon its ERK-mediated phosphorylation.

BDNF stimulates Ca 2+ oscillation frequency in melanotrope cells of Xenopus laevis: Contribution of IP3-receptor-mediated release of intracellular Ca 2+ to gene expression

Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating the animal’s skin color adaptation through secretion of α-melanophore-stimulating hormone (α-MSH). To fulfill this function optimally, the melanotrope cell undergoes plastic changes in structure and secretory activity in response to changed background light conditions. Xenopus melanotrope cells display Ca 2+ oscillations that are thought to drive α-MSH secretion and gene expression. They also produce brain-derived neurotrophic factor (BDNF), which stimulates in an autocrine way the biosynthesis of the α-MSH precursor, pro-opiomelanocortin (POMC). We have used this physiological adaptation mechanism as a model to investigate the role of BDNF in the regulation of Ca 2+ kinetics and Ca 2+-dependent gene expression. By dynamic video imaging of isolated cultured melanotropes we demonstrated that BDNF caused a dose-dependent increase in Ca 2+ oscillation frequency up to 64.7 ± 2.3% of control level. BDNF also induced a transient Ca 2+ peak in Ca 2+-free medium, which was absent when calcium stores were blocked by thapsigargin and 2-aminoethoxydiphenyl borate, indicating that BDNF stimulates acute release of Ca 2+ from IP 3-sensitive intracellular Ca 2+ stores. Moreover, we show that thapsigargin inhibits the expression of BDNF transcript IV (by 61.1 ± 28.8%) but does not affect POMC transcript. We conclude that BDNF mobilizes Ca 2+ from IP 3-sensitive intracellular Ca 2+ stores and propose the possibility that the resulting Ca 2+ oscillations selectively stimulate expression of the BDNF gene.

Physiological manipulation of cellular activity tunes protein and ultrastructural profiles in a neuroendocrine cell

To study in vivo the dynamics of the biosynthetic and secretory processes in a neuroendocrine cell, we use the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis. The activity of these cells can be simply manipulated by adapting the animal to a white or a black background, resulting in inactive and hyperactive cells respectively. Here, we applied differential display proteomics and field emission scanning electron microscopy (FESEM) to examine the changes in architecture accompanying the gradual transition of the inactive to the hyperactive melanotrope cells. The proteomic analysis showed differential expression of neuroendocrine secretory proteins, endoplasmic reticulum (ER)-resident chaperones, and housekeeping and metabolic proteins. The FESEM study revealed changes in the ultrastructure of the ER and Golgi and the number of secretory granules. We conclude that activation of neuroendocrine cells tunes their molecular machineries and organelles to become professional secretors.

Expression and physiological regulation of BDNF receptors in the neuroendocrine melanotrope cell of Xenopus laevis

Brain-derived neurotrophic factor (BDNF) and α-melanophore-stimulating hormone (α-MSH) are co-sequestered in secretory granules in melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis. α-MSH is responsible for pigment dispersion in dermal melanophores during the process of black-background adaptation. BDNF-production in melanotrope cells is increased by placing animals on a black background, and BDNF acts as an autocrine stimulatory factor on the melanotrope cells. However, the repertoire of possible neurotrophin receptors of the melanotrope is unknown. In this study we have established the expression of full length TrkB (TrkB.FL), truncated TrkB (TrkB.T) and p75 NTR receptors in the Xenopus neurointermediate lobe by RT-PCR. In situ hybridization reveals the presence of TrkB.FL mRNA and p75 NTR mRNA in melanotrope cells. Quantitative RT-PCR shows that in animals on a black background the amounts of TrkB.T and p75 NTR mRNA are about three times higher than in white background-adapted animals. We suggest that the amount of p75 NTR sets the sensitivity of the melanotrope cells for the stimulatory action of BDNF during physiological adaptation to background light intensity.

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.

Transgene expression of prion protein induces crinophagy in intermediate pituitary cells

The cellular form of the prion protein (PrP(C)) is a plasma membrane-anchored glycoprotein whose physiological function is poorly understood. Here we report the effect of transgene expression of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP-PrP(C)) specifically in the neuroendocrine intermediate pituitary melanotrope cells of Xenopus laevis. In the transgenic melanotrope cells, the level of the prohormone proopiomelanocortin (POMC) in the secretory pathway was reduced when the cells were (i) exposed for a relatively long time to the transgene product (by physiologically inducing transgene expression), (ii) metabolically stressed, or (iii) forced to produce unfolded POMC. Intriguingly, although the overall ultrastructure was normal, electron microscopy revealed the induction of lysosomes taking up POMC secretory granules (crinophagy) in the transgenic melanotrope cells, likely causing the reduced POMC levels. Together, our results indicate that in neuroendocrine cells transgene expression of PrP(C) affects the functioning of the secretory pathway and induces crinophagy.

Calcium Influx through Voltage‐Operated Calcium Channels Is Required for Proopiomelanocortin Protein Expression in Xenopus Melanotropes

Melanotrope cells of Xenopus laevis generate transitory increases in intracellular Ca(2+), known as Ca(2+) oscillations. These oscillations arise from the influx of Ca(2+) through voltage-operated Ca(2+) channels (VOCCs). Such oscillations are the driving force for secretion of a-melanophore-stimulating hormone (alpha-MSH) from the cell. The influx of Ca(2+) through VOCCs initiates the mobilization of intracellular Ca(2+) to generate a Ca(2+) wave. The function of the Ca(2+) wave in the melanotrope is unknown, but its presence in the nucleus suggests a role in the regulation of gene expression, perhaps that of proopiomelanocortin (POMC), the precursor protein for alpha-MSH. To determine the possible function of Ca(2+) waves in Xenopus melanotropes, we addressed whether functional VOCCs, which are an established requirement for both secretion and Ca(2+) wave initiation, are also required to maintain POMC gene expression.

Neuronal, Neurohormonal, and Autocrine Control of Xenopus Melanotrope Cell Activity

Amphibian pituitary melanotropes are used to investigate principles of neuroendocrine translation of neural input into hormonal output. Here, the steps in this translation process are outlined for the melanotrope cell of Xenopus laevis, with attention to external stimuli, neurochemical messengers, receptor dynamics, second-messenger pathways, and control of the melanotrope secretory process. Emphasis is on the pathways that neurochemical messengers follow to reach the melanotrope. The inhibitory messengers, dopamine, gamma-aminobutyric acid, and neuropeptide Y, act on the cells by synaptic input from the suprachiasmatic nucleus, whereas the locus coeruleus and raphe nucleus synaptically stimulate the cells via noradrenaline and serotonin, respectively. Autoexcitatory actions are exerted by acetylcholine, brain-derived neurotrophic factor (BDNF), and the calcium-sensing receptor. At least six messengers released from the pituitary neural lobe stimulate melanotropes in a neurohormonal way: corticotropin-releasing hormone, thyrotropin-releasing hormone, BDNF, urocortin, mesotocin, and vasotocin. They all are produced by the magnocellular nucleus and coexist in various combinations in two types of neurohemal axon terminal. Most of the relevant receptors of the melanotropes have been elucidated. Apparently, the neural lobe has a dominant role in activating melanotrope secretory activity. The intracellular mechanisms translating the various inputs into cellular activities like biosynthesis and secretion constitute the adenylyl cyclase-cAMP pathway and Ca(2+) in the form of periodic changes of the intracellular Ca(2+) concentration, known as Ca(2+) oscillations. It is proposed that the pattern of these oscillations encodes specific regulatory information and that it is set by first messengers that control, for example, via G proteins and cAMP-related events, specific ion channel-mediated events in the membrane of the melanotrope cell.

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.

Activity-dependent dynamics of coexisting brain-derived neurotrophic factor, pro-opiomelanocortin and alpha-melanophore-stimulating hormone in melanotrope cells of Xenopus laevis.

Brain-derived neurotrophic factor (BDNF) is involved as an autocrine factor in the regulation of the secretory activity of the neuroendocrine pituitary melanotrope cells of Xenopus laevis. We studied the subcellular distribution of BDNF in Xenopus melanotropes using a combination of high-pressure freezing, cryosubstitution and immunoelectron microscopy. Presence of BDNF, pro-opiomelanocortin (POMC) and alpha-melanophore-stimulating hormone (alphaMSH) within melanotrope secretory granules was studied by triple-labelling immunoelectron microscopy. In addition, intracellular processing of BDNF was investigated by quantifying the number of immunogold particles in different stages of secretory granule maturation, in animals adapted to black or white background light conditions. The high-pressure freezing technique provides excellent preservation of both cellular ultrastructure and antigenicity. BDNF coexists with POMC and alphaMSH within secretory granules. BDNF-immunoreactivity increases along the secretory granule maturation axis (i.e. from electron-dense, via moderately electron-dense, to electron-lucent secretory granules). Immature, low immunoreactive, electron-dense secretory granules are assumed to contain mainly or even exclusively proBDNF. Strongly immunoreactive electron-lucent secretory granules represent the mature granule stage in which proBDNF has been processed to mature BDNF. Furthermore, in moderately electron-dense secretory granules, immunoreactivity is markedly (+79%) higher in black-adapted than in white-adapted animals, indicating that stimulation of melanotrope cell activity by the black background condition speeds up processing of BDNF from its precursor in this granule stage. It is concluded that, in the Xenopus melanotrope, BDNF biosynthesis and processing occur along the secretory granule maturation axis, together with that of POMC-derived alphaMSH, and that the environmental light condition not only controls the biosynthesis and secretion of BDNF and of POMC end-products, but also regulates the rate of their intragranular processing.

Expression and characterization of the extracellular Ca(2+)-sensing receptor in melanotrope cells of Xenopus laevis.

The extracellular Ca(2+)-sensing receptor (CaR) is expressed in many different organs in various species, ranging from mammals to fish. In some of these organs, this G protein-coupled receptor is involved in the control of systemic Ca(2+) homeostasis, whereas in other organs its role is unclear (e.g. in the pituitary gland). We have characterized the CaR in the neuroendocrine melanotrope cell of the intermediate pituitary lobe of the South African clawed toad Xenopus laevis. First, the presence of CaR mRNA was demonstrated by RT-PCR and in situ hybridization. Then it was shown that activation of the CaR by an elevated extracellular Ca(2+) concentration and different CaR-activators, including L-phenylalanine and spermine, stimulates both Ca(2+) oscillations and secretion from the melanotrope. Furthermore, it was revealed that activation of the receptor stimulates Ca(2+) oscillations through opening of voltage-operated Ca(2+) channels in the plasma membrane of the melanotropes. Finally, it was shown that the CaR activator L-phenylalanine could induce the biosynthesis of proopiomelanocortin in the intermediate lobe. Thus, in this study it is demonstrated that the CaR is present and functional in a defined cell type of the pituitary gland, the amphibian melanotrope cell.

TRH signal transduction in melanotrope cells of Xenopus laevis

TRH is a neuropeptide that activates phospholipase C and, when acting on secretory cells, usually induces a biphasic response consisting of a transitory increase in secretion (due to IP 3 mobilization of Ca 2+ from intracellular stores), followed by a sustained plateau phase of stimulated secretion (by protein kinase C-dependent influx of extracellular Ca 2+ through voltage-operated Ca 2+ channels). The melanotrope cell of the amphibian Xenopus laevis displays a unique secretory response to TRH, namely a broad transient but no sustained second phase, consistent with the observation that TRH induces a single Ca 2+ transient rather than the classic biphasic increase in [Ca 2+] i . The purpose of the present study was to determine the signal transduction mechanism utilized by TRH in generating this Ca 2+ signaling response. Our hypothesis was that the transient reflects the operation of only one of the two signaling arms of the lipase (i.e., either IP 3-induced mobilization of internal Ca 2+ or PKC-dependent influx of external Ca 2+). Using video-imaging microscopy it is shown that the TRH-induced Ca 2+ transient is dramatically attenuated under Ca 2+-free conditions and that thapsigargin has no noticeable effect on the TRH-induced transient. These observations indicate that an IP 3-dependent mechanism plays no important role in the action of TRH. PKC also does not seem to be involved because an activator of PKC did not induce a Ca 2+ transient and an inhibitor of PKC did not affect the TRH response. Experiments with a bis-oxonol membrane potential probe showed that the TRH response also does not underlie a PKC-independent mechanism that would induce membrane depolarization. We conclude that the action of TRH on the Xenopus melanotrope does not rely on the classical phospholipase C-dependent mechanism.

New aspects of signal transduction in the Xenopus laevis melanotrope cell

Light and temperature stimuli act via various brain centers and neurochemical messengers on the pituitary melanotrope cells of Xenopus laevis to control distinct subcellular activities such as the biosynthesis, processing, and release of α-melanophore-stimulating hormone (αMSH). The melanotrope signal transduction involves the action of a large repertoire of neurotransmitter and neuropeptide receptors and the second messengers cAMP and Ca 2+. Here we briefly review this signaling mechanism and then present new data on two aspects of this process, viz. the presence of a stimulatory β-adrenergic receptor acting via cAMP and the egress of cAMP from the melanotrope upon a change of αMSH release activity.

Evidence that brain-derived neurotrophic factor acts as an autocrine factor on pituitary melanotrope cells of Xenopus laevis.

We have investigated the physiological regulation and functional significance of brain-derived neurotrophic factor (BDNF) in the endocrine melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis, which can adapt its skin color to the light intensity of its environment. In black-adapted animals, melanotrope cells produce and release alpha-melanophore-stimulating hormone (alpha-MSH). In white-adapted animals, the activity of melanotrope cells is inhibited by neuronal input. Using Western blotting and immunocytochemistry at the light and electron microscopical level, we have detected both the BDNF precursor and the mature BDNF protein in Xenopus melanotrope cells. In situ hybridization and RT-PCR revealed the presence of BDNF mRNA in the pituitary pars intermedia, indicating that BDNF is synthesized in the melanotropes. Real-time quantitative RT-PCR showed that levels of BDNF mRNA in melanotrope cells are about 25 times higher in black- than in white-adapted animals. Although there is no difference in the amount of stored mature BDNF, the amount of BDNF precursor protein is 3.5 times higher in melanotropes of black-adapted animals than in those of white-adapted animals. These data indicate that BDNF mRNA expression and BDNF biosynthesis are up-regulated in active melanotrope cells. Because immunoelectron microscopy showed that BDNF is located in melanotrope secretory granules, BDNF is probably coreleased with alpha-MSH via the regulated secretory pathway. Superfusion and (3)H-amino acid incorporation studies demonstrated that BDNF stimulates the release of alpha-MSH and the biosynthesis of its precursor protein, POMC. Our results provide evidence that BDNF regulates the activity of Xenopus melanotrope cells in an autocrine fashion.

Evidence that Brain-Derived Neurotrophic Factor Acts as an Autocrine Factor on Pituitary Melanotrope Cells of Xenopus laevis

Evidence that Brain-Derived Neurotrophic Factor Acts as an Autocrine Factor on Pituitary Melanotrope Cells of Xenopus laevis

Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis

In the present study, we examined the amphibian Xenopus laevis as a model for stable transgenesis and in particular targeted transgene protein expression to the melanotrope cells in the intermediate pituitary. For this purpose, we have fused a Xenopus proopiomelanocortin (POMC) gene promoter fragment to the gene encoding the reporter green fluorescent protein (GFP). The transgene was integrated into the Xenopus genome as short concatemers at one to six different integration sites and at a total of one to ∼20 copies. During early development the POMC gene promoter fragment gave rise to GFP expression in the total prosencephalon, whereas during further development expression became more restricted. In free-swimming stage 40 embryos, GFP was found to be primarily expressed in the melanotrope cells of the intermediate pituitary. Immunohistochemical analysis of cryosections of brains/pituitaries from juvenile transgenic frogs revealed the nearly exclusive expression of GFP in the intermediate pituitary. Metabolic labelling of intermediate and anterior pituitaries showed newly synthesized GFP protein to be indeed primarily expressed in the intermediate pituitary cells. Hence, stable Xenopus transgenesis with the POMC gene promoter is a powerful tool to study the physiological role of proteins in a well-defined neuroendocrine system and close to the in vivo situation.

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes.

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.

Minimal model for intracellular calcium oscillations and electrical bursting in melanotrope cells of Xenopus laevis.

A minimal model is presented to explain changes in frequency, shape, and amplitude of Ca2+ oscillations in the neuroendocrine melanotrope cell of Xenopus Laevis. It describes the cell as a plasma membrane oscillator with influx of extracellular Ca2+ via voltage-gated Ca2+ channels in the plasma membrane. The Ca2+ oscillations in the Xenopus melanotrope show specific features that cannot be explained by previous models for electrically bursting cells using one set of parameters. The model assumes a KCa-channel with slow Ca2+-dependent gating kinetics that initiates and terminates the bursts. The slow kinetics of this channel cause an activation of the Kca-channel with a phase shift relative to the intracellular Ca2+ concentration. The phase shift, together with the presence of a Na+ channel that has a lower threshold than the Ca2+ channel, generate the characteristic features of the Ca2+ oscillations in the Xenopus melanotrope cell.

Action Currents Generate Stepwise Intracellular Ca2+Patterns in a Neuroendocrine Cell

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.