Role Of Intracellular Calcium Research Articles

Role of intracellular calcium and protein kinases in the activation of hepatic Na+/taurocholate cotransport by cyclic AMP.

Glucagon and dibutyryl cyclic AMP (Bt2cAMP) stimulate Na+/taurocholate (TC) cotransport and increase the intracellular Ca2+ concentration ([Ca2+]i) of hepatocytes. Whether the effect of cAMP is mediated via increases in [Ca2+]i, cAMP-dependent protein kinase (PKA), and/or protein kinase C (PKC) was investigated in this study. TC uptake and [Ca2+]i were determined in isolated rat hepatocytes using [14C]TC and the fluorescent dye quin-2, respectively. Bt2cAMP, forskolin, and 8-bromo-cAMP stimulated Na(+)-dependent, but not Na(+)-independent TC uptake. Bt2cAMP increased the maximal rate of Na+/TC cotransport without affecting the apparent Km. Increases in TC uptake and [Ca2+]i by Bt2cAMP were inhibited in hepatocytes preloaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (MAPTA) or preincubated with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate (TMB8). Calmodulin antagonists inhibited Bt2cAMP-induced increases in TC uptake, but not [Ca2+]i. Other Ca(2+)-mobilizing agents (thapsigargin, vasopressin, phenylephrine, and ionomycin) increased [Ca2+]i but failed to stimulate TC uptake, indicating that an increase in [Ca2+]i alone is not a sufficient stimulus for TC uptake. However, increases in TC uptake by 1 and 10 microM Bt2cAMP were further increased by thapsigargin, indicating a permissive role for Ca2+/calmodulin. Bt2cAMP-induced increases in TC uptake and [Ca2+]i were inhibited by known inhibitors of PKA and by an activator of PKC, but they remained unaffected by a specific inhibitor of PKC. Unlike thapsigargin, vasopressin inhibited Bt2cAMP-induced increases in TC uptake. Taken together these results indicate that stimulation of hepatic Na+/TC cotransport by cAMP 1) is mediated via PKA; 2) is potentiated, but not mediated, by Ca2+/calmodulin-dependent processes; and 3) may be down-regulated by PKC.

Osmolarity-stimulated urea transport in rat terminal IMCD: role of intracellular calcium

We showed previously that both increasing osmolality by adding NaCl or manitol (hyperosmolarity) or adding vasopressin can stimulate urea permeability in rat terminal inner medullary collecting ducts (IMCD). Vasopressin acts via adenosine 3',5'-cyclic monophosphate (cAMP), but the mechanism by which hyperosmolarity acts is unknown. To study the mechanism, we determined the effect of varying osmolality (with NaCl) on two potential second messenger systems, i.e., cAMP and intracellular calcium. There was no significant difference in cAMP production among tubules incubated at 290, 490, 690, or 890 mosmol/kg. In contrast, cAMP did increase significantly after vasopressin (10(-8) M) addition. Intracellular calcium increased significantly when osmolality was increased from 290 to 490 mosmol/kg in the absence of vasopressin. To examine whether changes in intracellular calcium affect urea permeability, we added thapsigargin (and removed bath calcium) while maintaining osmolality at 290 mosmol/kg. Both intracellular calcium and urea permeability increased significantly. Next, we buffered intracellular calcium by pretreatment with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM). BAPTA completely blocked the increase in urea permeability occurring when osmolality was increased from 290 to 490 mosmol/kg, but did not block the increase in urea permeability occurring when vasopressin (10(-8) M) was added. In summary, 1) hyperosmolarity increases intracellular calcium, but has no effect on cAMP accumulation; 2) thapsigargin increases intracellular calcium and urea permeability; and 3) BAPTA blocks the hyperosmolarity-stimulated increase in urea permeability, but not vasopressin-stimulated urea permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective chemotaxis and calcium response in localized juvenile periodontitis neutrophils.

Localized juvenile periodontitis (ljp) is an early onset form of periodontal disease characterized by unique localization to first molars and incisors and a high prevalence of neutrophil abnormalities, particularly chemotaxis. The intracellular transduction mechanisms that follow receptor-ligand coupling on the neutrophil surface and lead to chemotaxis are not clearly established. Chemotaxis and phagocytosis are modulated by a variety of receptors and involve several activation pathways; the role of intracellular calcium as a presumptive second messenger and mediator of these events is well established. The putative effector mechanisms for the chemotactic receptor of neutrophils also include the possible activation of a phospholipase, protein kinase C, methyltransferase, or adenylate cyclase. In normal neutrophils, a phosphoinositide pathway initiated by phospholipase C, which results in the activation of protein kinase C via diacylglycerol and the generation of IP3, has been implicated. In order to better understand the stages of neutrophil transduction, fluorescent probes were used to monitor neutrophil calcium changes. Chlorotetracycline (CTC) was used as an indirect probe of intracellular membrane-bound pool of calcium stores, and Quin-2 was used to monitor cytosolic free calcium levels of FMLP stimulated normal and LJP neutrophils. The results indicate that the early phase of the calcium response affiliated with the release of intracellularly sequestered calcium appears intact in LJP neutrophils, as the CTC fluorescence changes were similar to control values. The second phase of the calcium response, associated with membrane channel activation and an influx of extracellular calcium, appeared compromised in the neutrophils of the LJP population.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nifedipine and ruthenium red on the contractile function and oxidative metabolism of the myocardium

The role of intra- and extracellular calcium in beta-adrenergic regulation of rat's heart have been shown. It was found that oxidative metabolism of resistant and non-resistant animals to hypoxia is under control of 2 different intracellular calcium pools. The role of intracellular calcium is more important in non-resistant to hypoxia rats.

The Role of Intracellular Calcium and pH during Fertilization and Egg Activation in the Hydrozoan Phialidium

A calcium transient occurs at fertilization in the eggs of the hydrozoans Mitrocomella and Phialidium. The eggs of Phialidium have an intracellular pH (pH i) of 7.6-7.95. There is no increase in pH i following fertilization. Both calcium ionophere and ammonia treatments activate Phialidium eggs. Calcium ionophore causes a calcium transient without changing pH i. Ammonia concentrations of 10-20 m M at pH 8 cause a rise in pH i but no detectable calcium transient. Both activating agents can (1) block subsequent fertilization, (2) initiate cell cycle events such as DNA synthesis, nuclear envelope breakdown, chromosome condensation, and cleavage-related events, and (3) initiate voltage-dependent calcium channel function. While calcium ionophore treatment invariably elicits all of these manifestations of egg activation, ammonia sometimes induces only the induction of voltage-dependent calcium channel function. Oocytes were also treated with calcium ionophore or ammonia at different stages during their maturation. Ammonia treatment did not induce egg activation when applied at any stage during maturation; however, calcium ionophore initiated voltage-dependent calcium channel function when oocytes were treated after germinal vesicle breakdown. Ionophore treatment during maturation did not render these eggs unfertilizable or initiate cell cycle events. These experiments show that elevation of [Ca 2+] with ionophore and increasing pH i with ammonia may activate these eggs through different pathways, that specific maturational events must occur before they can do so, and that the activation of voltage-dependent calcium channel function can be dissociated from other egg activation events.

Role of intracellular calcium in NI-35-evoked collapse of neuronal growth cones.

A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, inhibits regeneration of lesioned neuronal fiber tracts in vivo and growth of neurites in vitro. Growth cones of cultured rat dorsal root ganglion neurons arrested their growth and collapsed when exposed to liposomes containing NI-35. Before morphological changes, the concentration of free intracellular calcium ([Ca2+]i) showed a rapid and large increase in growth cones exposed to liposomes containing NI-35. Neither an increase in [Ca2+]i nor collapse of growth cones was detected in the presence of antibodies to NI-35. Dantrolene, an inhibitor of calcium release from caffeine-sensitive intracellular calcium stores, protected growth cones from collapse evoked by NI-35. Depletion of these caffeine-sensitive intracellular calcium stores prevented the increase in [Ca2+]i evoked by NI-35. The NI-35-evoked cascade of intracellular messengers that mediates collapse of growth cones includes the crucial step of calcium release from intracellular stores.

Volume regulation in guinea pig outer hair cells and the role of intracellular calcium.

Single outer hair cells (OHCs) isolated from the guinea pig cochlea showed a regulatory volume decrease (RVD) after the initial cells swelling despite continued exposure to a hypotonic solution. The accompanying change of the intracellular Ca2+ concentration ([Ca2+]i) in the OHCs was investigated using the Ca(2+)-sensitive dye fura-2. Hyposmotic activation led to a [Ca2+]i increase which was accompanied by cell shortening and swelling. In a nominally Ca(2+)-free solution, however, [Ca2+]i was not significantly increased during hyposmotic activation although shortening and swelling of the OHCs were observed. These results suggest that the increase in [Ca2+]i during hyposmotic activation is mainly based on an influx of extracellular Ca2+.

A Topic Revisited: the Unique and Shared Properties of Neuronal Growth Cones That Enable Navigation and Specific Pathfinding

One of the primary responsibilities of the neuronal growth cone is its role of guiding an elongating neurite to its proper target. The events during extension and pathfinding require that the neuronal growth cone responds to particular molecules in the environment by making appropriate changes in the behaviour of the growth cone. This paper discusses the role of intracellular calcium as a signalling system mediating these responses. The specific roles of the growth cone filopodia in acting as antennae in advance of the motile growth cone are discussed in the context of calcium mediated pathfinding events.

Role of intracellular calcium in renal proximal tubule cell volume regulation.

Osmoregulatory Ca2+ signaling in hypotonic solutions was studied with videometric techniques in 158 proximal renal tubules isolated from the teleost Carassius auratus. Absence of extracellular Ca2+, hypoxia (23 mmHg), or NaCN (3 mM) did not alter regulatory volume decreases (RVD). Nevertheless, decrements of intracellular Ca2+ via the A23187 ionophore or after intracellular Ca2+ chelation with indo-1/AM (5 microM) inhibited RVD. In tubules depleted of Ca2+, RVD could only be fully elicited when intracellular Ca2+ pulses were given within 1 min after hypotonic stimulation. While inhibition of Ca2+ release from the endoplasmic reticulum (ER) with 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 50 microM) blunted RVD, some of its effects could be reversed with the anion carrier tributyltin (1 microM). Dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP, 0.5 and 1.0 mM) and forskolin (0.25 mM) also impeded RVD; however, their effects could be partially reversed with the K+ ionophore gramicidin (0.5 microM). In conclusion, in Carassius auratus proximal renal tubule cells, RVD is activated by an intracellular Ca2+ signal that likely emanates from the ER and not from the extracellular media or the mitochondrial Ca2+ pool. Ca2+ activation of a cAMP-modulated osmoregulatory K+ channel appears to play an important role.

Role of intracellular calcium in hydrogen peroxide-induced renal tubular cell injury.

Both reactive oxygen metabolites and calcium have been implicated in ischemic and toxic renal tubular cell injury. However, the role of calcium in oxidant injury to renal tubular cells has not been previously examined. In the present study we examined the role of intracellular free Ca2+ ([Ca2+]i) in H2O2-mediated injury to LLC-PK1 cells, a renal tubular epithelial cell line. H2O2 induced a significant rise in [Ca2+]i within 1 min after exposure of cells to 5 mM H2O2, with a sustained rise in [Ca2+]i during the course of experiments, reaching a value of 1.3 microM at 60 min (n = 10). The rise in [Ca2+]i preceded sublethal cell injury as measured by [3H]adenine release or irreversible cell injury as determined by trypan blue exclusion. Buffering [Ca2+]i with quin-2 (50 microM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) was highly protective against the H2O2-induced cell injury. For example, at 120 min after exposure of cells to 5 mM H2O2, irreversible cell injury was reduced from 45 +/- 8 to 9 +/- 1% (n = 3) by quin-2. The acetoxymethyl ester of quin-2 (quin-2/AM) and BAPTA/AM did not interfere with the trypan blue exclusion assay or scavenge H2O2. Preventing mobilization of Ca2+ from intracellular storage sites using 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 10(-4) M) significantly reduced the rise in [Ca2+]i and thus prevented H2O2-mediated cytotoxicity to LLC-PK1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of calcium in rat oligodendrocyte injury and repair.

The role of intracellular calcium in oligodendrocyte injury is investigated using cultured rat oligodendrocytes. Calcium ionophores A23187 and ionomycin mimic both complement and perforin attack, causing oligodendrocyte lysis at concentrations which do not lyse other glia. Membrane vesiculation, the mechanism by which oligodendrocytes resist and recover from complement and perforin attack, is also induced by A23187. Oligodendrocytes are more susceptible to complement attack in the presence of a calmodulin inhibitor (W7), which also inhibits vesiculation. These results imply that calmodulin is involved in membrane repair from complement attack, and indicate that changes in intracellular calcium play an important yet paradoxical role in the oligodendrocyte response to injury, dictating both susceptibility and cellular recovery.

Mechanisms of acute ischemic contractile failure of the heart. Role of intracellular calcium.

Mechanisms of acute ischemic contractile failure of the heart. Role of intracellular calcium.

Flunarizine protects sensory and sympathetic neurones from death after NGF withdrawal and axotomy: a possible role of intracellular calcium in determining trophic factor dependence

Flunarizine protects sensory and sympathetic neurones from death after NGF withdrawal and axotomy: a possible role of intracellular calcium in determining trophic factor dependence

Neomycin, an inhibitor of phosphoinositide hydrolysis, inhibits the resumption of bovine oocyte spontaneous meiotic maturation.

The possibility that the intracellular signals generated upon phosphoinositide hydrolysis are involved in regulating bovine oocyte spontaneous meiotic resumption was investigated. Oocytes were mass-harvested and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin in the presence or absence of neomycin (an inhibitor of phosphoinositide hydrolysis) or phorbol myristate acetate (an activator of protein kinase C). The role of intracellular calcium was examined by preloading with BAPTA/AM (a calcium chelator) prior to culture. Meiotic maturation was scored cytogenetically. 1) Neomycin induces an irreversible inhibition of germinal vesicle breakdown which does not exceed 60% and is apparent at concentrations of 5 mM or above. Progression of meiosis past metaphase I is inhibited at concentrations of 2.5 mM or above. The full effect of neomycin is only apparent if it is presented to the oocytes within 3 h of follicular release, although germinal vesicle breakdown is not observed until 9 h culture under control conditions. 2) PMA alone has negligible effect on germinal vesicle breakdown, but it acts synergistically with 2 mM IBMX to inhibit this process. PMA has a dual effect on the progression of meiosis past metaphase I: 1 nM PMA has a stimulatory effect while 1 microM PMA blocks the ability of oocytes to reach anaphase I or beyond. These observations are not found with a non-tumor-promoting phorbol ester. 3) Spontaneous meiotic resumption is not significantly affected in the absence of added exogenous calcium. However, oocytes preloaded with BAPTA/AM exhibit a dose-dependent inhibition of germinal vesicle breakdown, even in the presence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of growth cone behavior by calcium

Few structures within the nervous system have received more attention than the neuronal growth cone. Initially named by Ramon y Cajal (1890) who recognized its dynamics and importance in histological sections, the growth cone plays essential roles in the development, repair, and modification of neuronal circuitry. Growth cones have a diverse repertoire of behaviors (Fig. 1). These behaviors underlie the more complex processes of neurite elongation, pathfinding, and selective synaptogenesis. Thus, a fundamental concern of developmental neurobiology is the definition of environmental and intracellular cues that regulate growth cone behavior. In this essay, we will consider the role of intracellular calcium in the regulation of growth cone behaviors. The impetus for envisaging such a role was provided by the elucidation of the calcium hypothesis of the control of secretion (Douglas, 1974, 1976) and synaptic transmission (Katz and Miledi, 1965; Katz, 1969; Llinas, 1979). Accordingly, we put forward a working hypothesis that stated, “if [intracellular] calcium falls below an optimal level, or rises significantly above it, growth cone motility and neurite outgrowth are inhibited” (Rater et al., 1988a).

Role of intracellular calcium in volume regulation by rabbit medullary thick ascending limb cells

Previous studies demonstrated that Ca2(+)-activated K+ channels in luminal membrane of rabbit medullary thick ascending limb cells (MTAL) are activated on exposure of the cells to hyposmotic solutions [J. Taniguchi and W. B. Guggino. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F347-F352, 1989]. In this study, we investigated the mechanism of activation of Ca2(+)-activated K+ channels in MTAL cells exposed to hyposmotic solutions. MTAL cells swell in hyposmotic medium and regulate volume back toward the starting volume. This regulatory volume decrease (RVD) is inhibited at high medium K+ concentrations or by presence of quinine or Ba2+ in extracellular medium, suggesting involvement of K+ channels. Measurements of intracellular Ca2+ concentrations with fura-2 show that intracellular Ca2+ rises in hyposmotic solutions and that this rise does not occur in absence of extracellular Ca2+. Nifedipine and verapamil also inhibit rise in intracellular Ca2+. Decreasing intracellular Ca2+ by removal of external Ca2+ in presence of EDTA or by chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibits RVD. We conclude that hypotonic solutions activate K+ efflux probably via K+ channels and Ca2+ influx via a nifedipine- and verapamil-sensitive pathway. Lowering intracellular Ca2+ removes the ability of MTAL cells to regulate volume in hyposmotic solutions.

Cytosolic free Ca 2+ in daunorubicin and vincristine resistant ehrlich ascites tumor cells : Drug accumulation is independent of intracellular Ca 2+ changes

The possible role of intracellular calcium on daunorubicin (DNR) accumulation in wild-type (EHR2) and multi-drug resistant (MDR) Ehrlich ascites tumor cell subline was investigated. DNR accumulation was not enhanced either by increasing the concentration of cellular calcium with the calcium ionophore ionomycin nor by chelating the cytosolic free Ca 2+ by the membrane permeable Ca 2+-buffering agents BAPTA or MAPTAM. No effect was observed in the presence of extremely low extracellular calcium concentration that prevent transmembrane calcium influx or when the cells were calcium depleted using EGTA and ionomycin. Using the fluorescent Ca 2+ indicator fura-2 it is further shown that both drug-resistant daunorubicin (EHR2/DNR+) and vincristine (EHR/VCR+) sublines had lower (50–80 nM) concentration of cytosolic free calcium ([Ca 2+] i) compared to their corresponding wild-type parenteral tumors (140–180 nM). In calcium free medium, however, no significant difference was found, all cell lines having a [Ca 2+] i of 60–80 nM. Furthermore, the total amount of Ca 2+ released to the cytosol with 10 μM ionomycin and 5 mM EGTA was 3–4-fold higher in EHR2 than in EHR2/ DNR+ or EHR2/VCR+. Mobilization of Ca 2+ with 1 μM ionomycin was almost identical in the presence and absence of Ca 2+ in the extracellular medium in EHR2 as well as in EHR2/DNR+ suggesting that the increase in [Ca 2+] i is mainly due to discharge of Ca 2+ from intracellular stores. Furthermore, the total cell calcium [Ca 2+] i concentration was slightly higher in EHR2/DNR+ and EHR2/VCR+ cells compared to EHR2. Incubation of the cells with the Ca 2+-channel blocker verapamil or the intracellular Ca 2+-antagonist TMB-8 causes depression of the Ca 2+-response in terms of rise in [Ca 2+] i caused by ionomycin. Sorcin, a major calcium-binding protein ( M r, 22 kDa), is shown to be overproduced in EHR2/DNR+ cells. The overproduction of this protein in resistant cells may be related to the difference in the intracellular calcium observed in this study. Thus, though handling of Ca 2+ is different in wild-type and MDR cell lines, our data suggest that calcium is not involved directly in drug transport processes and the level of Ca 2+ per se have no influence on drug accumulation.

Morphological changes associated with furazolidone-induced cardiomyopathy: Effects of digoxin and propranolol

The purpose of the present study was to examine light microscopic data qualitatively as well as quantitatively from an animal model of dilated cardiomyopathy in the turkey. A previous study reported the gross cardioprotective effect of propranolol and the lack of cardioprotection with digoxin in furazolidone-induced cardiomyopathy. It was therefore important to define whether the response of the myocardium to therapeutic interventions differed from the structural responses seen in their absence. In furazolidone-treated birds with resultant cardiac dilation there was myocyte hypertrophy, enlargement of nuclei and reorientation of subepicardial myocardial fibres. Similar changes were noted in birds receiving both furazolidone and digoxin. However, birds receiving propranolol, a non-selective beta-receptor antagonist, and furazolidone did not demonstrate a hypertrophic response or reorientation of fibres. These data indicate that propranolol maintained myocardial morphology and morphometry and thus prevented the structural sequelae of the disease, which is a better achievement of therapy than simple arrest of the progress. A role for intracellular calcium is implied. The cardioprotective effect seen with beta blockade suggests that membrane related events may lead to the contractile dysfunction that results in dilation and cardiac hypertrophy.

The role of intracellular calcium in arrhythmias in early ischaemia : D.G. Allen, J.A. Lee. Department of Physiology, University College London, Gower Street, London WC1E 6BT

The role of intracellular calcium in arrhythmias in early ischaemia : D.G. Allen, J.A. Lee. Department of Physiology, University College London, Gower Street, London WC1E 6BT

Actin assembly in electropermeabilized neutrophils: role of intracellular calcium.

Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium [( Ca2+]i) in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca2+ ionophores can be known to induce actin assembly, the following observations suggest that an increased [Ca2+]i is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2(+)-free medium, depleted of internal Ca2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2+]i, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 microM free Ca2+, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]i beyond the resting level by direct addition of CaCl2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]i resulted in spontaneous actin assembly. To reconcile these findings with the actin-polymerizing effects of Ca2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]i. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]i, but rather that elevated [Ca2+]i facilitates actin disassembly, an effect possibly mediated by Ca2(+)-sensitive actin filament-severing proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.