New approaches in the study of stimulus-secretion coupling in anterior pituitary cells.
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Amino Acid Sequence↗
Biomedical subjects
W T Mason
Publications and source records attributed to W T Mason.
Extracellular pH determines the rate of Ca2+ entry into Madin-Darby canine kidney-focus cells.
We investigated the relationship between intracellular Ca2+ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca2+ concentration (Ca2+i). Ca2+i and intracellular pH (pHi) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca2+ influx from the extracellular space into the cell was determined with the Mn2+ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca2+i oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca2+i oscillations. On the contrary, changes of extracellular pH value strongly affected Ca2+i oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pHi changes were less than 0.1 pH units. However, experiments with the Mn2+ quenching technique revealed that extracellular alkalinization significantly reduced Ca2+ entry from the extracellular space. Large increases of Ca2+i triggered by the blocker of the cytoplasmic Ca(2+)-ATPase, thapsigargin, had no effect on pHi. We conclude: intracellular Ca2+ homeostasis in MDCK-F cells is pH dependent. pH controls Ca2+ homeostasis mainly by effects on the level of Ca2+ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rap changes of Ca2+i.
Animals↗
Cell transformation induces a cytoplasmic Ca2+ oscillator in Madin-Darby canine kidney cells.
Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca2+ concentration ([Ca2+]i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP3)-sensitive Ca2+ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca(2+)-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca2+]i showed that Ca2+ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca2+ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca2+ release through transformation. Oscillations are primarily due to the activity of an InsP3-sensitive cytosolic Ca2+ oscillator.
Animals↗
Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells.
Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca2+ oscillations from an inositol 1,4,5-triphosphate-sensitive cytoplasmic Ca2+ store. In this study, Ca2+ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca2+ video imaging and the Fura-2/Mn2+ quenching technique. Oscillations were dependent on extracellular Ca2+ concentration and were inhibited by extracellularly applied La3+, Co2+ and Ni2+. Depolarization of the cell membrane with high K+ concentrations and the L-type Ca2+ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca2+ channels. Mn2+ quenching experiments disclosed significant Ca2+ influx into MDCK-F cells. The rate of this influx was constant between Ca2+ spikes, but markedly increased during the spontaneous Ca2+ spikes. Similar transient increases in Ca2+ entry could be mimicked by agents triggering intracellular Ca2+ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca2+ permeability permitting Ca2+ entry into the cytoplasm. The rate of Ca2+ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca2+ concentration.
Animals↗
Different G proteins are involved in the biphasic response of clonal rat pituitary cells to thyrotropin-releasing hormone.
In rat anterior pituitary tumour cells (GH3/B6) thyrotropin-releasing hormone (TRH) elicits a biphasic response. First, a release of intracellularly stored Ca2+ induces a hyperpolarization of the cell. Second, a depolarization thought to be induced by a reduction of the inward-rectifying K+ current (KIR) causes an increase in action potential frequency and a plateau-like increase in [Ca2+]i. It has been proposed that the two phases are induced by the actions of inositol 1,4,5-trisphosphate (InsP3) and protein kinase C (PKC), respectively, but we demonstrate here that PKC is not responsible for the second phase increase in [Ca2+]i and suggest that the pathways diverge at the level of receptor and G protein coupling. Both phases of the TRH response were insensitive to pertussis toxin, but cholera toxin (CTX) selectively affected the second phase. After CTX pretreatment cells had a high spontaneous spiking frequency and smaller KIR amplitude. In these cells TRH failed to increase the action potential frequency after the first phase hyperpolarization, elicited only a transient peak increase in [Ca2+]i with no plateau phase and could only slightly reduce KIR. These effects of CTX are not mediated by its ability to increase cAMP via activation of GS, as increased cAMP levels neither inhibit KIR nor prevent its reduction by TRH. In addition, inhibition of protein kinase A activation did not block the second phase increase in [Ca2+]i induced by TRH, suggesting that the CTX-sensitive G protein mediating the second phase of the TRH response is not GS.
Action Potentials↗
Study of stimulus-secretion coupling in single cells using antisense oligodeoxynucleotides and patch-clamp techniques to inhibit specific protein expression.
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Base Sequence↗
Extracellular ATP activates calcium entry and mobilization via P2U-purinoceptors in rat lactotrophs.
Extracellular ATP has been previously shown to activate calcium signalling in pituitary cell populations [1] but the particular cell types involved have not been identified. We used video imaging of Fura-2 loaded into single rat pituitary cells and identified as lactotrophs to study the effects of extracellular ATP on [Ca2+]i. ATP does not permeabilize the cells as shown by exclusion of propidium iodide. ATP causes two types of calcium transients in lactotrophs. The most common response is a rapid increase in [Ca2+]i that decays slowly and is terminated by washout of ATP. This type of response is also seen in calcium-free medium, demonstrating mobilization of calcium stores dependent upon the presence of the agonist. ATP also stimulates calcium entry as detected by Mn(2+)-quenching of Fura-2. ATP in Mg(2+)-free medium and ATP gamma S are effective agonists suggesting ATP4- is the active form. The presence of P2-purinoceptors is apparent because ATP, ADP and AMP increase [Ca2+]i in decreasing order of potency and adenosine has no effect. ATP-induced calcium transients are reduced by the P2-purinoceptor antagonists suramin and quinidine. UTP is equipotent with ATP and defines the receptor subtype as P2U. We conclude that ATP4- acts on rat lactotrophs via P2U-purinoceptors to elevate [Ca2+]i from intracellular and extracellular sources.
Adenosine Triphosphate↗
Rab3 proteins: key players in the control of exocytosis.
Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic.
Animals↗
Effects of pituitary adenylate cyclase-activating polypeptide in the pituitary: activation of two signal transduction pathways in the gonadotrope-derived alpha T3-1 cell line.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is thought to play a hypophysiotropic role, but little is known of the identity of PACAP-stimulated cells in the pituitary, the nature of the PACAP receptors on specific cell types, and the effector systems for these receptors. Here we describe the effects of PACAP in alpha T3-1 cells, a gonadotrope-derived cell line. In these cells, PACAP38 causes concentration-dependent increases in cAMP accumulation (EC50, 3 nM), [3H]inositol phosphate ([3H]IP) production (EC50, 20 nM), and the cytosolic Ca2+ concentration. The Ca2+ response is biphasic and is sustained only in Ca(2+)-containing medium. Intact alpha T3-1 cells possess a single class of [125I]PACAP27-binding sites (Kd, 3.3 nM; binding capacity, 35 fmol/10(6) cells). The rank orders of potencies for stimulation of cAMP and [3H]IP production and for inhibition of [125I] PACAP27 binding by three related peptides are identical (PACAP38 = PACAP27 > > vasoactive intestinal peptide). In addition to stimulation of LH release from primary cultures of rat pituitary cells and [3H] IP accumulation in alpha T3-1 cells, PACAP38 synergizes with low GnRH concentrations in the production of these effects. Moreover, long term exposure to PACAP38 stimulates [3H]thymidine incorporation and increases steady state levels of the gonadotropin alpha-subunit in alpha T3-1 cells. We conclude that alpha T3-1 cells possess type I PACAP receptors which mediate the observed effector system responses, and demonstration of the effects of PACAP on this gonadotrope-derived cell line provides further evidence that gonadotropes are direct targets for PACAP action. The data imply that stimulation of phospholipase-C by PACAP is responsible (at least in part) for the observed increase in cytosolic Ca2+, which, in turn, probably mediates the effects of PACAP on LH release. We suggest, however, that in gonadotropes, the effects of PACAP on cell replication and gonadotropin synthesis may prove more important than the peptide's modest effects on LH release.
Animals↗
rab3-peptide stimulates exocytosis from mast cells via a pertussis toxin-sensitive mechanism.
Synthetic peptides of the putative effector domain of members of the rab3 gene family of small GTP-binding proteins have been shown to have potent actions on vesicular transport and exocytosis [1,2]. Here, we use similar rab3-effector domain peptides to study their role in intracellular signalling in mast cells. We find that rab3-like peptides stimulate exocytosis and decrease cyclic 3',5'-AMP levels in these cells when applied extracellularly. Cells pretreated with pertussis toxin (PtX) to selectively uncouple alpha i/alpha o type G proteins from their biological activators, however, did not respond to rab3 peptides. rab3-like peptides also induce a Ca2+ transient in mast cells. These observations provide evidence for functional coupling between an effector domain peptide sequence of rab3 protein and a PtX-sensitive G protein substrate.
Amino Acid Sequence↗
Inhibition of Rab3B expression attenuates Ca(2+)-dependent exocytosis in rat anterior pituitary cells.
Low-molecular-weight GTP-binding proteins (small G proteins) of the Rab family have been proposed to act as central regulators of vesicular traffic, and proteins of the Rab3 subfamily (Rab3A, B, C and D) are thought to be associated with membrane vesicles or granules undergoing exocytotic fusion with the plasma membrane. Rab3A is highly expressed in brain, whereas Rab3B is the major form found in rat anterior pituitary gland. We report here that antisense oligonucleotides against Rab3B, introduced into pituitary cells using the whole-cell patch clamp technique, specifically and reversibly block expression of Rab3B. We find that calcium-dependent exocytosis is inhibited without affecting endocytosis. Antisense oligonucleotides directed against Rab3A have no effect. Our results indicate that Rab3B is likely to be a key intracellular signalling molecule which can control exocytosis downstream of other calcium-dependent processes in anterior pituitary cells.
Animals↗
Calcium signaling in neurosecretory terminals and pituicytes.
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Animals↗
Roles of phospholipase C and Ca(2+)-ATPase in calcium responses of single, fibrinogen-bound platelets.
Fura-2-loaded platelets were immobilized on fibrinogen, and cytosolic free calcium concentration ([Ca2+]i) was measured by video imaging of the fluorescence signal. In the immobilized, single platelets, ADP and low doses of thrombin evoked repetitive spikes of [Ca2+]i, whereas higher thrombin concentrations gave elevated plateaus in [Ca2+]i. Stimulation of the cells with thrombin after the addition of ADP changed the frequency of spiking, but not the maximal levels of [Ca2+]i reached. In suspensions of platelets, ADP and low doses of thrombin evoked transient elevation of myo-inositol phosphates, suggesting that the initiation of spiking was due to stimulation of phospholipase C. In platelet suspensions, the Ca(2+)-ATPase inhibitor thapsigargin (TG) evoked a gradual rise in [Ca2+]i, which was potentiated by preactivation of the platelets and inhibited by prostaglandin E1 and nitroprusside. In single platelets, TG induced a sudden increase in [Ca2+]i after a long but variable delay, followed by a phase of slow oscillations. The effects of preactivation with ADP were 2-fold: the delay time before the response to TG was shortened and the maximal level of [Ca2+]i reached with TG was higher than the level of the preceding Ca2+ spikes. Apparently, Ca2+ responses induced by the inhibition of Ca(2+)-ATPases are potentiated by prior elevation of [Ca2+]i and reduced by substances that inhibit agonist-evoked increases in [Ca2+]i. The data point to a mechanism of Ca(2+)-induced Ca2+ release and the presence of TG-sensitive and TG-insensitive Ca2+ stores in platelets. Rapid spiking may involve both pools, whereas the latter alone may account for the slow TG-evoked oscillations.
Adenosine Diphosphate↗
Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy.
The properties of pH and calcium homeostasis have been investigated in single bovine lactotrophs by the use of the fluorescent indicators 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF) and fura-2 respectively. A method of simultaneous recording from both dyes loaded in the same cell was used. Despite slight crosstalk between the two dyes, physiologically relevant information about the interrelationship between pH and calcium homeostasis was obtained. Three types of interactions were recorded. First, an increase in calcium due to the discharge of intracellular stores by thyrotropin-releasing hormone resulted in no change in cytosolic pH. Secondly, alkalinization by the addition of a weak base, NH4Cl, induced a large transient (around 1000 nM) and a small (a few tens of nanomoles per liter) sustained increase in cytosolic calcium. The former is partly due to release from agonist-sensitive stores. Thirdly, upon the removal of NH4Cl the cytoplasm became acidic, which induced a release of calcium from intracellular stores in some cells. In addition we demonstrate that the recovery from acid load is sensitive to extracellular Na+, suggesting the presence of Na+/H+ exchange mechanisms in bovine lactotrophs. Interestingly we have also found that, at rest, removal of Na+ from the bathing medium results in a decrease in resting [Ca2+]i, paralleled by a reduction in pHi. This suggests a role for Na+/H+ exchange in determining resting [Ca2+]i.
Ammonium Chloride↗
Dynorphin 1-17 delays the vasopressin induced mobilization of intracellular calcium in cultured astrocytes from the rat neural lobe.
Opioid peptides are present in nerve terminals in the rat neural lobe where they partially coexist with vasopressin. Morphological findings suggest that these neuropeptides are released onto pituicytes, which is in agreement with a possible role for the pituicyte in oxytocin and vasopressin release from the neural lobe. Pituicytes in culture respond to vasopressin with a mobilization of calcium from intracellular stores. In the present study this vasopressin induced increase in intracellular free calcium levels was both delayed and decreased by pre-exposure to dynorphin 1-17, while dynorphin 1-17 by itself did not affect basal calcium levels. All effects of dynorphin 1-17 could be blocked with naloxone. The present results suggest that opioid receptors are present on pituicytes and are coupled to a second messenger pathway by which opioid peptides may inhibit inositol phosphate dependent calcium mobilization by other neuropeptides, such as vasopressin.
Animals↗
Control of growth hormone secretion at the single cell level.
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Animals↗
Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts.
Cell-matrix interactions have been shown to play an important role in regulating cell function and behaviour. In bone, where calcified matrix formation and resorption events are required to be in dynamic equilibrium, regulation of adhesive interactions between bone cells and their matrix is critical. The present study focuses on the osteoclast, the bone resorbing cell, as well as integrins, which are cell surface adhesion receptors that mediate osteoclast attachment to bone matrix. In osteoclasts, the most abundant integrin receptor is the vitronectin receptor (VNR, alpha v beta 3). The objective of the study was to investigate changes in intracellular calcium, a regulator of osteoclast function, following addition of peptides that bind integrins, in particular the alpha v beta 3 form of the vitronectin receptor (VNR), which is highly expressed in osteoclasts. The study demonstrated a unique spatial localisation of the calcium signal in response to cell membrane receptor occupancy by integrin ligands in rat osteoclasts. Addition of peptides with the Arg-Gly-Asp (RGD) sequence such as BSP-IIA, GRGDSP and GRGDS to rat osteoclasts evoked an immediate increase in free calcium ion concentration [Ca2+]i, localised to the nuclei and to the thin cytoplasmic skirt. These responses were inhibited by F11, a monoclonal antibody to the rat integrin beta 3 chain, as well as echistatin, a snake venom shown to colocalise with the alpha v chain in osteoclasts, suggesting that the calcium signal is mediated by the alpha v beta 3 form of VNR.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Arginine vasopressin mobilises intracellular calcium via V1-receptor activation in astrocytes (pituicytes) cultured from adult rat neural lobes.
An extremely close association exists between the membranes of the neurosecretory endings and the resident astrocytes (pituicytes) of the neurohypophysis. Indeed, synaptoid contacts involving neurosecretory vesicle-containing axons contacting pituicytes have been observed, suggesting pituicytes as targets of the products released from neurosecretory axons. We have investigated the effects of various neural lobe peptides on pituicytes in primary culture from adult neurohypophyses. Using Fura-2 loaded cells and dynamic ratio imaging, we have determined that arginine vasopressin (AVP) or V1- but not V2-receptor agonists, mobilise pituicyte intracellular Ca2+ ([Ca2+]i) in the absence of extracellular Ca2+. AVP was consistently effective at concentrations of 10 nM or higher in elevating [Ca2+]i by 200-1000 nM. These responses could be blocked by V1-antagonists and were shown to be associated with accumulation of phosphoinositides. Oxytocin was also found to mobilise [Ca2+]i but was effective only at higher concentrations than for AVP. Oxytocin-evoked [Ca2+]i elevations were also blocked by V1-antagonists. Raising [K+]0 was ineffective in changing [Ca2+]i suggesting that these cells lack voltage-gated Ca2+ channels. We conclude that pituicytes possess V1-receptors, activation of which mobilises [Ca2+]i, possibly functioning to initiate a Ca(2+)-activated K+ conductance which could contribute to further depolarisation of secretory terminals and facilitate exocytosis.
Animals↗