Transient Increase In Cytoplasmic Ca2 Research Articles

Oxytocin is involved in cellular signaling within the retina

BackgroundOxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G‐protein coupled receptor. We have recently localized OXTR to the retinal pigment epithelium (RPE) and OXT to the adjacent cone photoreceptors.ObjectiveWe sought to characterize the signaling mechanism of OXTR in the RPE and explore the downstream effects of OXT, focusing on the regulation of inwardly rectifying K+ channel Kir7.1.MethodsCa2+ response to OXT was measured in cultured human fetal RPE cells (hfRPE) using Fura‐2 AM in the presence of 2‐APB and nifedipine, pharmacological inhibitors of Ca2+ signaling pathways. HEK‐293 cells where used for the stable expression of human OXTR. Cellular signaling was visualized using live cell fluorescence imaging following transient expression of PH‐GFP and PKC‐GFP, monitors of GPCR metabolites PIP2 and DAG. Whole cell patch clamp electrophysiology was performed on HEK‐OXTR cells transfected with GFP‐fused Kir7.1 as well as freshly isolated mouse RPE cells to study Kir7.1 channel function.ResultsOXT treatment of RPE cells in culture resulted in a transient increase in cytoplasmic Ca2+ that was reduced by 95% in the presence of the IP3R antagonist, 2‐APB (P < 0.0001). Upon bathing the cells in Ca2+‐free extracellular solution or nifedipine, the Ca2+ response to OXT was not inhibited, although amplitude was reduced. Time to the rise in [Ca2+]i was not altered but recovery was slowed down with time constants (τ) of 0.35 ± 0.02 (r2 =.99), 0.34 ± 0.03 (r2 =.98), and 0.20 ± 0.005 (r2=.99) min for Ca2+‐free, nifedipine, and Ringer's solution, respectively. We also demonstrate that OXTR activation blunted Kir7.1 channel current. In isolated mouse RPE, we observed an average 61.81 ± 4.77 % decrease in K+ inward current amplitude at ‐160mV and an average 11.4 ± 3.2 mV depolarization in resting membrane potential by OXT.ConclusionWe propose that OXTR stimulates a mobilization of intracellular Ca2+, independent of extracellular Ca2+, through 2nd messengers coupled to OXTR/G‐protein in the RPE. This OXT‐OXTR signaling in the RPE cell also integrated the parallel modulation of the Kir7.1 channel. Kir7.1 channel is responsible for controlling RPE resting membrane potential and can influence retinal health and vision. We suggest that novel OXT‐OXTR signaling pathways in the outer retina will be of fundamental importance for eye development, health and visual function.Support or Funding InformationNIH EY024995, UnityPoint Health Meriter Foundation, Pediatrics, Graduate School, School of Medicine and Public Health

The N-methyl-D-aspartate-evoked cytoplasmic calcium increase in adult rat dorsal root ganglion neuronal somata was potentiated by substance P pretreatment in a protein kinase C-dependent manner

The involvement of substance P (SP) in neuronal sensitization through the activation of the neurokinin-1-receptor (NK1r) in postsynaptic dorsal horn neurons has been well established. In contrast, the role of SP and NK1r in primary sensory dorsal root ganglion (DRG) neurons, in particular in the soma, is not well understood. In this study, we evaluated whether SP modulated the NMDA-evoked transient increase in cytoplasmic Ca2+ ([Ca2+]cyt) in the soma of dissociated adult DRG neurons. Cultures were treated with nerve growth factor (NGF), prostaglandin E2 (PGE2) or both NGF+PGE2. Treatment with NGF+PGE2 increased the percentage of N-methyl-d-aspartate (NMDA) responsive neurons. There was no correlation between the percentage of NMDA responsive neurons and the level of expression of the NR1 and NR2B subunits of the NMDA receptor or of the NK1r. Pretreatment with SP did not alter the percentage of NMDA responsive neurons; while it potentiated the NMDA-evoked [Ca2+]cyt transient by increasing its magnitude and by prolonging the period during which small- and some medium-sized neurons remained NMDA responsive. The SP-mediated potentiation was blocked by the SP-antagonist ([D-Pro4, D-Trp7,9]-SP (4–11)) and by the protein kinase C (PKC) blocker bisindolylmaleimide I (BIM); and correlated with the phosphorylation of PKCε. The Nk1r agonist [Sar9, Met(O2)11]-SP (SarMet-SP) also potentiated the NMDA-evoked [Ca2+]cyt transient. Exposure to SP or SarMet-SP produced a rapid increase in the labeling of phosphorylated-PKCε. In none of the conditions we detected phosphorylation of the NR2B subunit at Ser-1303. Phosphorylation of the NR2B subunit at Tyr1472 was enhanced to a similar extent in cells exposed to NMDA, SP or NMDA+SP, and that enhancement was blocked by BIM. Our findings suggest that NGF and PGE2 may contribute to the injury-evoked sensitization of DRG neurons in part by enhancing their NMDA-evoked [Ca2+]cyt transient in all sized DRG neurons; and that SP may further contribute to the DRG sensitization by enhancing and prolonging the NMDA-evoked increase in [Ca2+]cyt in small- and medium-sized DRG neurons.

Role of presynaptic and postsynaptic IP3‐dependent intracellular calcium release in long‐term potentiation in sympathetic ganglion of the rat

In the rat superior cervical ganglion, a form of long term potentiation (LTP) can be elicited by a brief high frequency stimuli applied to the preganglionic nerve. Cumulative evidence shows that a transient increase in cytoplasmic Ca²+ concentration is essential for the generation of the ganglionic LTP. Calcium influx and calcium release from intracellular calcium stores contribute to LTP. However, the differential role of presynaptic and postsynaptic calcium signaling has not been established. Herein, by using heparin, a membrane-impermeant inositol trisphosphate receptor (IP3R) blocker, we explored the contribution of presynaptic and postsynaptic IP3-sensitive calcium stores to the ganglionic LTP. The LTP was produced by a conditioning train of 40 Hz for 3 s. We analyzed the effects of heparin on the posttetanic potentiation: PTP magnitude and PTP time constant, and on two parameters that describe the LTP: LTP decay time (elapsed time required by the potentiated response to fall to 20% above the basal value) and LTP extent (the integral of the potentiated response). Heparin (100 and 200 μg/ml) was loaded in the preganglionic, the postganglionic, or in both nerves. We found that in all tested conditions heparin significantly decreased LTP but practically did not affect PTP. The preganglionic and postganglionic inhibitory effects of heparin were not additive. De-N-sulfated heparin, an ineffective IP3R blocker, had no effect on LTP, but abolished the heparin blocking effect. Data suggest that presynaptic and postsynaptic IP3-dependent intracellular calcium release equally contribute to ganglionic LTP, supporting our proposal of a trans-synaptic mechanism for LTP.

Multiplex Kinase Signaling Modifies Cardiac Function at the Level of Sarcomeric Proteins

The transient change in tension development and length in a working cardiac myocyte during the heart beat reflects the integrated effects of kinases in signaling cascades regulating mechanisms controlling the dynamics and intensity of a transient increase in cytoplasmic Ca2+ as well as the responsiveness of the sarcomeres to Ca2+. Kinases modifying regulatory membrane proteins represent a major mechanism for controlling the coupling of transmembrane voltage to the release of Ca2+ into the cytoplasm that triggers contraction by binding to TnC2 and for regulating dynamics of the return of Ca2+ to the diastolic state by membrane transporters and exchangers (1). Binding of Ca2+ to TnC triggers a strong reaction of sarcomere thick filament cross-bridges with the thin filament actins and the promotion of force development and shortening (2, 3). Sarcomeres are not passive responders to these transient changes in cytoplasmic Ca2+. Protein-protein interactions downstream of Ca2+-TnC are subject to functionally significant modifications by signaling cascades that modify the number and kinetics of actin-cross-bridge reactions (Fig. 1). FIGURE 1. Kinases affecting sarcomeric proteins. Major substrates for these kinases are illustrated in a region of overlap between thin actin-containing and thick myosin-containing filaments. Also shown is a portion of the network of proteins at the Z-disk, ... I focus here on control mechanisms at the level of the sarcomere and on kinases immediately upstream of sarcomeric protein substrates. Major substrates are (i) thin filament proteins TnI, TnT, and Tm, which are important in transducing the Ca2+-TnC signal (4, 5); (ii) MyBP-C (6) and RLC (7), which control the radial movement of cross-bridges from the thick filament backbone; and (iii) titin, a giant third filament controlling diastolic tension as well as length-dependent radial movement of cross-bridges (8, 9). Detailed discussion of how phosphorylation modifies the function of these proteins has been reviewed elsewhere (2, 4–9). In general, phosphorylation of thin filament proteins controls sarcomere Ca2+ sensitivity, kinetics of Ca2+ binding to TnC (related to dynamics of relaxation), and the number and kinetics of cross-bridges reacting with the thin filament (related to levels and rates of rise and fall of tension). Phosphorylations of MyBP-C and RLC control Ca2+ sensitivity and rates of contraction/relaxation by modifying the local concentration of cross-bridges at the interface with actins. MyBP-C may also interact with and affect thin filament activation. Cardiac but not skeletal isoforms of titin contain phosphorylation sites within a unique sequence, located in the elastic segment. Phosphorylation of a unique cardiac titin reduces passive tension (8). To appreciate the potential role of how kinases modify sarcomeric function, it is important to consider the working cardiac myocyte operating in an environment influenced by immediate prevailing mechanical (load and length), neural, endocrine, autocrine, and paracrine control mechanisms and by the short- and long-term history of this environment. Beat-to-beat control mechanisms, which occur, for example, as hemodynamic load rises with exercise or falls with rest, are related to the immediate prevailing regulatory mechanisms. Mechanisms occurring over the time scale of hours, days, and longer are related to growth and remodeling in response to chronic changes in load or chemical environment as occur with sustained bouts of exercise, hypertension, or ischemia. Kinases and phosphorylations play a significant role in compensation and adaptation to beat-to-beat and chronic changes in hemodynamic load. However, maladaptive kinase activation may induce remodeling and phosphorylations of sarcomeric proteins with cardiovascular disorders, leading to heart failure (10, 11).

Calcineurin is required to release Xenopus egg extracts from meiotic M phase

Fertilization induces a transient increase in cytoplasmic Ca2+ concentration in animal eggs that releases them from cell cycle arrest in the second meiotic metaphase. In frog eggs, Ca2+ activates Ca2+/calmodulin-activated kinase, which inactivates cytostatic factor, allowing the anaphase-promoting factor to turn on and ubiquitinate cyclins and securin, which returns the cell cycle to interphase. Here we show that the calcium-activated protein phosphatase calcineurin is also important in this process. Calcineurin is transiently activated after adding Ca2+ to egg extracts, and inhibitors of calcineurin such as cyclosporin A (ref. 8) delay the destruction of cyclins, the global dephosphorylation of M-phase-specific phosphoproteins and the re-formation of a fully functional nuclear envelope. We found that a second wave of phosphatase activity directed at mitotic phosphoproteins appears after the spike of calcineurin activity. This activity disappeared the next time the extract entered M phase and reappeared at the end of mitosis. We surmise that inhibition of this second phosphatase activity is important in allowing cells to enter mitosis, and, conversely, that its activation is required for a timely return to interphase. Calcineurin is required to break the deep cell cycle arrest imposed by the Mos-MAP (mitogen-activated protein) kinase pathway, and we show that Fizzy/Cdc20, a key regulator of the anaphase-promoting factor, is an excellent substrate for this phosphatase.

Excitotoxic Injury to Mitochondria Isolated from Cultured Neurons

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by approximately 10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 +/- 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 +/- 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 +/- 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca(2+)-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.

Isoflurane Reduction of Carbachol-evoked Cytoplasmic Calcium Transients Is Dependent on Caffeine-sensitive Calcium Stores

Many muscarinic functions are relevant to anesthesia, and alterations in muscarinic activity affect the anesthetic/analgesic potency of various drugs. Volatile anesthetics have been shown to depress muscarinic receptor function, and inhibition of the muscarinic signaling pathway alters the minimal alveolar anesthetic concentration of inhaled anesthetics. The purpose of this investigation was to determine in a neuronal cell which source of Ca2+ underlying the carbachol-evoked transient increase in cytoplasmic Ca2+ was reduced by isoflurane. Experiments were performed at 37 degrees C on continuously perfused monolayers of human neuroblastoma SH-SY5Y cells using Fura-2 as the cytoplasmic Ca2+ indicator. Carbachol (1 mm) was applied to evoke a transient increase in cytoplasmic Ca2+. Isoflurane (1 mm) reduces the carbachol-evoked transient increase in cytoplasmic Ca2+, and this isoflurane action is eliminated when the cells are continuously stimulated with 200 mm KCl or pretreated with 10 mm caffeine or 200 microm ryanodine. Isoflurane reduction of the carbachol-evoked transient increase in cytoplasmic Ca2+ requires full caffeine-sensitive Ca2+ stores and Ca2+ release from the caffeine-sensitive stores through the ryanodine-sensitive Ca2+ release channels. The results indicate that isoflurane interferes with a muscarinic Ca2+ signaling through a mechanism downstream from the muscarinic receptors.

AMPA/kainate and NMDA-like glutamate receptors at the chromatophore neuromuscular junction of the squid: role in synaptic transmission and skin patterning.

Glutamate receptor types were examined at the chromatophore synapses of the squids Alloteuthis subulata and Loligo vulgaris, where nerve-induced muscle contraction causes chromatophore expansion. Immunoblotting with antibody raised against a squid AMPA receptor (sGluR) demonstrated that AMPA/kainate receptors are present in squid skin. Application of l-glutamate evoked chromatophore muscle contractions in both ventral and dorsal skins, while NMDA was only active on a subpopulation of dorsal chromatophores. In dorsal skin, neurotransmission was partly blocked by either AMPA/kainate receptor antagonists (CNQX and DNQX) or NMDA receptor antagonists (AP-5 and MK-801) or completely blocked by simultaneous application of both classes of antagonists. In isolated muscle fibres, ionophoretic application of l-glutamate evoked fast inward CNQX- and DNQX-sensitive currents with reversal potentials around +14 mV and a high conductance to Na+. In fibres from dorsal skin only, a slower outward glutamate-sensitive current appeared at positive holding potentials. At negative potentials, currents were potentiated by glycine or by removing external Mg2+ and were blocked by AP-5 and MK-801. Glutamate caused a fast, followed by a slow, transient increase in cytoplasmic Ca2+. The slow component was increased in amplitude and duration by glycine or by lowering external Mg2+ and decreased by AP-5 and MK-801. In cells from ventral skin, no 'NMDA-like responses' were detected. Thus, while AMPA/kainate receptors mediated fast excitatory synaptic transmission and rapid colour change over the whole skin, activation of both AMPA/kainate and NMDA-like receptors in a subpopulation of dorsal chromatophores prolonged the postsynaptically evoked Ca2+ elevation causing temporally extended colour displays with behavioural significance.

Endothelial permeability for macromolecules. Mechanistic aspects of pathophysiological modulation.

The endothelium separates blood from tissue and actively regulates the exchange between these compartments. Throughout the body, this one-cell-thick lining of blood vessels extends over several thousand square meters. It harbors many receptors and functions that actively regulate the extravasation of nutrients, solutes, hormones, macromolecules, and leukocytes. If the barrier function of the endothelium does not perform appropriately, leakage of macromolecules occurs, causing edema formation and exposure of the interstitium to high concentrations of plasma constituents. In severe cases of vascular leakage, platelets will adhere to the exposed subendothelium and red blood cells may even accumulate in the interstitium. Vascular leakage may be desirable for recruiting plasma proteins, such as complement factors during infection, but it can become life-threatening, particularly when it occurs in the lungs or pericardium or when it causes hypovolemic shock. Furthermore, frequent or prolonged leakage may impair proper functioning of the affected tissue. In recent years, considerable progress has been made in understanding the molecular processes that contribute to the regulation of endothelial permeability. With the exception of the liver, adrenal, and bone marrow sinusoids, in which the endothelium has rather large pores, the endothelium constitutes a selective barrier between blood and tissue. In principle, this barrier consists of the EC monolayer and its matrix. Macromolecules can cross the endothelial barrier in three ways: (1) between the cells, through cell junctions (paracellular); (2) through the EC, via pores (diaphragms or fused vesicles); and (3) transcellularly, via shuttling vesicles and specific receptors. It is generally believed that the charge and compactness of the endothelial matrix (both glycocalyx and basal membrane) contribute additionally to the selectivity of the endothelial barrier toward molecules of different size and charge. Electron microscopic evidence strongly suggests that in tissue capillaries, hormones and macromolecules are shuttled across the endothelial barrier via vesicles, in …

Cytoplasmic Hydration Triggers a Transient Increase in Cytoplasmic Ca2+ Concentration in Nitella flexilis

Cytoplasmic Hydration Triggers a Transient Increase in Cytoplasmic Ca2+ Concentration in Nitella flexilis

Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly.

Cell locomotion and changes in cell structure initiated by binding of extracellular ligands to transmembrane receptors are generally accompanied by a transient increase in cytoplasmic Ca2+ concentration, [Ca2+], and by synthesis and hydrolysis of phosphorylated phosphatidylinositol lipids, or polyphosphoinositides (PPls). These two potential signals to cyto­ skeleton are not necessarily immediate consequence of receptor stimu­ lation, but are themselves modulated by upstream signals that include GTPases, protein kinases, ion channels, and probably other agents (reviewed in 118). Cell motility and changes in morphology also require spatially and temporally coordinated changes in mechanical properties of cell cortex, and these changes are brought about by transitions between gel (solid) and sol (liquid) states of cytoskeleton (32, 119, 120). A link between Ca2+ and phospholipids, on one hand, and changes in cell rigidity, on other, was apparent enough to suggest nearly 40 years ago (52) that the rigidity of cortex depends on fact that it contains

Mitogenic signaling mechanisms of human cementum-derived growth factors.

Cementum-derived growth factor (CGF) is a M(r) 23,000 protein, which is sequestered in the mineralized matrix of tooth cementum. We have investigated the mitogenic signaling reactions induced by CGF using quiescent human gingival fibroblasts as target cells. Cells activated with CGF were compared with those treated with CGF plus epidermal growth factor (EGF) and other growth factors. CGF caused a transient increase in cytoplasmic Ca2+ concentration, and this was accompanied by enhancement of membrane protein kinase C activity, myelin basic protein and S6 kinase activities, inositol phosphate levels, and activation of c-fos and jun-B gene expression. Membranes obtained from cells activated with CGF contained several protein bands, which cross-reacted with antiphosphotyrosine antibody; however, proteins corresponding to a putative phosphorylated CGF receptor were not detected. DNA synthesis induced by CGF was inhibited by 65% in cells treated with pertussis toxin but only 25-29% in cultures exposed to H7 or 12-O-tetradecanoylphorbol-13-acetate; these values were different from those obtained when EGF, PDGF, or fetal bovine serum were used as mitogens. CGF and TGF-beta, but not EGF, caused an increase of PDGF-A chain mRNA expression 4 h after mitogen addition. However, while CGF was mitogenic for gingival fibroblasts, TGF-beta was not. Kinetics of DNA stimulation and experiments with anti-PDGF antibodies indicated that PDGF-A expression does not contribute significantly to CGF-induced DNA synthesis. When the stimulation of various signaling pathways induced by CGF and other growth factors was compared, the pattern of stimulation by CGF was different from other growth factors. The characteristic signaling reactions of CGF are likely to be important components of the mechanisms that regulate the formation and regeneration of cementum and adjacent connective tissues.

Interrelationship between growth factor-induced pH changes and intracellular Ca2+.

Many mitogens cause rapid changes in intracellular pH and Ca2+. We studied the patterns of pH and Ca2+ changes after exposure of murine fibroblasts to platelet-derived growth factor (PDGF), bombesin, phorbol 12-myristate 13-acetate (PMA), and the vasoactive peptide bradykinin. Intracellular pH and Ca2+ were measured by using the fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2. Three distinct patterns of intracellular pH change were observed. PDGF and bombesin caused a rapid (maximum change, less than 2 min) cytoplasmic acidification of 0.03 pH unit followed by a slower (5-10 min) alkalinization of approximately 0.11 pH unit above the resting pH of 6.88. PMA caused alkalinization without causing the early acidification. Bradykinin caused rapid acidification without the slower net alkalinization. Ionomycin also caused acidification without subsequent alkalinization. All acidification responses were amiloride resistant. Patterns of intracellular Ca2+ response were also determined for each agent. PDGF and bombesin caused a transient increase in cytoplasmic Ca2+ from a resting level of 85 +/- 12 nM to 190 +/- 12 nM within 2 min and return to baseline within 5 min. PMA caused no change in intracellular Ca2+. Bradykinin caused the most rapid (maximum response, less than 20 sec) increase in intracellular Ca2+. For each agonist, the Ca2+ transient could be blocked by buffering intracellular Ca2+ with quin-2. In Ca2+-buffered cells, PDGF, bombesin, bradykinin, and ionomycin failed to induce cellular acidification, but alkalinization responses to PDGF, bombesin, and PMA persisted. We propose that the transient acidification seen with PDGF, bombesin, and other agents is the result of increased intracellular Ca2+. However, growth factor-induced alkalinization via the Na+/H+ exchanger is independent of changes in Ca2+.