Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist

Calcium-mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2-loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline-induced Ca2+ signals. Gap junction blockade with 18 alpha-glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline-induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell-cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a 'pacemaker-like' intercellular spread of Ca2+ waves.     

Spiral intercellular calcium waves in hippocampal slice cultures

Complex patterns of intercellular calcium signaling occur in the CA1 and CA2 regions of hippocampal slice organotypic cultures from neonatal mice. Spontaneous localized intercellular Ca2+ waves involving 5-15 cells propagate concentrically from multiple foci in the stratum oriens and s. radiatum. In these same regions, extensive Ca2+ waves involving hundreds of cells propagate as curvilinear and spiral wavefronts across broad areas of CA1 and CA2. Ca2+ waves travel at rates of 5-10 mu m/s, are abolished by thapsigargin, and do not require extracellular Ca2+. Staining for astrocytes and neurons indicates that these intercellular waves occur primarily in astrocytes. The frequency and amplitude of Ca2+ waves increase in response to bath application of N-methyl-D-aspartate (NMDA) and decrease in response to removal of extracellular Ca2+ or application of tetrodotoxin. This novel pattern of intercellular Ca2+ signaling is characteristic of the behavior of an excitable medium. Networks of glial cells in the hippocampus may behave as an excitable medium whose spatial and temporal signaling properties are modulated by neuronal activity.     

Propagation of mechanically induced intercellular calcium waves via gap junctions and ATP receptors in rat liver epithelial cells

Mechanical stimulation was used to initiate Ca2+ waves in rat liver epithelial cells in order to ascertain the degree to which gap junctional intercellular communication (GJIC) is involved in communication of Ca2+ to adjacent cells and to assess alternative Ca2+ signaling pathways that may be present between these cells. In both WB-F344 cells, which show a high degree of GJIC, and WB-aB1 cells, which are GJIC deficient, mechanical stimulation of a single cell induced a Ca2+ wave which propagated away from the point of stimulation, across cell borders, to neighboring cells directly or indirectly in contact with the stimulated cell. In addition, the Ca2+ wave was transmitted to nearby isolated cells that exhibited no direct or indirect contact with the stimulated cell. Treatment of cells with 18beta-glycyrrhetinic acid, a compound that has been shown to block GJIC, did not significantly affect propagation of the Ca2+ wave. In contrast, treatment with suramin, a P2-purinergic receptor inhibitor, significantly reduced both the rate and the extent of Ca2+ wave propagation in WB-F344 cells and completely blocked its propagation in WB-aB1 cells. Cotreatment with suramin and glycyrrhetinic acid was found to completely block the mechanically induced Ca2+ wave in both cell lines. These studies indicate that mechanically induced cell injury in rat liver epithelial cells initiates signaling through at least two pathways, involving intercellular communication via gap junctions and extracellular communication via ATP activation of purinergic receptors.     

Plasma albumin induces calcium waves in rat cortical astrocytes

At the end of the 19th century, ectopic pregnancy became a surgical procedure. A century later, one third of ectopic pregnancies are treated medically. In the meantime, early detection of ectopic pregnancy became possible due to sensitive serum hCG and progesterone combined assays with transvaginal sonography and a knowledge of risk factors. Consequently, a nonsurgical approach appears to be an attractive alternative to surgery. Expectant management is recommended with a plateau or decreasing hCG and an initial level < or = 1.000 mIU/ml in asymptomatic women. Medical treatment by local or parenteral methotrexate is recommended in patients with clear evidence of an unruptured pregnancy in based on initial hCG and progesterone level, size of hemoperitoneum, ultrasound diameter of hematosalpinx and absence of clinical pain. Laparoscopy remains the gold standard but in prospective randomized trials between medical treatment and laparoscopy, in selected patients, the non-surgical approach appears to be equivalent with a similar reproductive performance.     

The effect of calcium channel blockers and calcium on methotrexate accumulation in rat hepatocytes

The effects of diltiazem (DIL), verapamil (VRP) and Ca2+ on the accumulation of methotrexate (MTX) were investigated in isolated rat hepatocytes. At the physiological 2 mM Ca2+, the calcium-channel blockers DIL (100 microM) and VRP (50 microM) significantly reduced the hepatocellular accumulation of MTX. By increasing the Ca2+ concentration to 7 mM control MTX levels (at 2 mM Ca2+) were restored with VRP, and resulted in MTX levels above the controls for DIL. Ca2+ at 7 mM significantly enhanced MTX accumulation in the hepatocyte suspensions after 60 min. The concentration time curves for MTX indicated that for the first 10 min influx was the dominating process. Dixon plot analysis of this uptake phase revealed Ki values of 140 microM for DIL and 75 microM for VRP. The data suggested that DIL was a non-competitive, and VRP a competitive inhibitor of MTX influx. Hence, the inhibitory effect on MTX accumulation mediated by DIL and VRP could be due to different mechanisms.     

Ultrastructural characteristics of intercellular contacts and bile canaliculi in neonatal rat hepatocytes in primary culture

The ultrastructure of the cellular contacts and bile canaliculi was examined in cultured neonatal (day 5) rat hepatocytes to elucidate the development of cellular polarity. A new scanning electron microscopic technique for cultured hepatocytes allowed a view of cell-cell attachment and the entire cell surface, including the underside on plastic dishes. At 3 h after plating, neonatal hepatocytes were shown to be round, with loss of the preferential localization of cell organelles. After 6 h of culture, the cells had become oblong; they were aggregated in groups of several cells and the cellular contacts were not as rigid or as straight as those in adult hepatocytes. Transmission electron microscopy showed the biliary functional polarity to be like that in vivo. On the undersurfaces of adjacent neonatal hepatocytes a hemicanalicular structure lined with microvilli was found, which probably corresponds to the ultrastructure of bile canaliculi in vivo. However, no canaliculi or orifices of bile channels were found in adult hepatocytes. These results suggest that in neonatal rat hepatocytes the formation of tight rigid cellular contacts was suppressed. Modulation of cell membranes appeared on the undersurfaces of neonatal hepatocytes in early culture stages. The differences in the development of cellular polarity could be caused by the proliferating activity of neonatal hepatocytes.     

Hormone-mediated calcium responses of rat and human hepatocytes. Study of multicellular systems by videomicroscopy

OBJECTIVES AND METHODS: Activation of hepatocyte hormonal receptors leads to the mobilization of intracellular Ca2+ which is thought to be an elaborate system for encoding hormonal messages. We studied hormone-induced calcium signals in freshly isolated multicellular systems of normal rat and human hepatocytes. Calcium signals were recorded by videomicroscopy after stimulation with noradrenaline, angiotensin II, and vasopressin. RESULTS: Calcium signals were highly organized in multiplets: the different hepatocytes responded to Ca(2+)-mobilizing hormones in a sequentially ordered manner, with a first, a second (doublets) and a third (triplets) responding cells. This pattern was an intrinsic feature of the multicellular systems, and seemed to be a result of a gradual heterogeneity of the sensitivity of the different cells, to the hormones. The stimulation of the same multiplet with two different agonists and the removal of the hormone during cell responses provides some evidence for the major role of hormonal receptors in this heterogeneity. CONCLUSIONS: Hormone responses in multicellular systems of rat and human hepatocytes are highly elaborate. The density of hormonal receptors could be the major determinant of the sequential pattern of Ca2+ responses. Hormonal receptors may be gradually distributed among the different cells of the multiplets in vitro and along the porto-centrilobular axis in situ.     

Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.     

Mechanism involved in initiation and propagation of receptor-induced intercellular calcium signaling in cultured rat astrocytes

The mechanisms involved in the initiation and the propagation of intercellular calcium signaling (calcium waves) were studied in cultured rat astrocytes. The analysis of calcium waves, induced either by mechanical stimulation or by focal application of ionomycin, indicated that initiation was dependent on the presence of external calcium. In addition, pharmacological experiments indicate that intercellular propagation required PLC activation, integrity of IP3-sensitive internal calcium stores, and functional gap junctions. An extracellular action of ATP or glutamate and participation of voltage-dependent Ca2+ channels were tested by using enzymatic degradation, receptor antagonists, and channel blockers, respectively. Because neither the speed of propagation nor the extent of the calcium waves was affected by these treatments, these alternate mechanisms were excluded from playing a role in intercellular calcium signaling. Biochemical assays and focal applications of several agonists (methoxamine, carbachol, glutamate) of membrane receptors to neurotransmitters and peptides (endothelin 1) demonstrated that their ability to trigger regenerative calcium waves depended on phospholipase C activity and inositol phosphate production. Thus, in rat astrocytes, initiation and propagation of calcium waves involve a sequence of intra- and intercellular steps in which phospholipase C, inositol trisphosphate, internal calcium stores, and gap junction channels play a critical role. The identification of these different events allows us to determine several targets at which the level of long-range signaling in astrocytes may be controlled.     

Inhibition of calcium influx during hypoxia/reoxygenation in primary cultured rat hepatocytes

Calcium has been demonstrated to play an important role in hepatocyte damage during ischemia/reperfusion phases. Calcium influx was determined in primary cultured rat hepatocytes submitted to a succession of warm hypoxia and reoxygenation phases in the presence of diltiazem, gallopamil and a Na+/H+ antiport inhibitor, HOE-694. Only diltiazem significantly inhibited calcium influx with higher potency after reoxygenation than after hypoxia only, suggesting a complex mechanism of action of diltiazem which could act on different physiological functions involved in Ca2+ invasion of hepatocytes after hypoxic insult.     

Uptake of tyramine by rat hepatocytes

Observations on the uptake of tyramine by hepatocytes indicate that the amine is taken up by simple diffusion and a transporter mediated system, with a Km of 39 microM and a Vmax of 270 pmol/min/10(5) cells. The carrier-mediated process is pH- and temperature-dependent and requires an activation energy of 12.9 kcal/mol. An overshoot uptake is achieved a few minutes after adding this amine to the cell suspension, suggesting that active transport is involved. This is supported by the finding that partial inhibition of the uptake can be induced by oligomycin, azide, cyanide and dinitrophenol. NO3-, SCN- and SO4(2-), which change the membrane potential significantly, and depress the transporter mediated uptake further, suggesting that the membrane potential is the driving force for the entry of this amine across hepatic membrane. Cysteine is essential for the normal carrier function; whereas, histidine, tryptophan, arginine and lysine do not directly deal with the activity of the carrier. Many substances, but not amino acids, H, M, and N receptor agonists, can inhibit the uptake of tyramine. It is possible that other amines can enter hepatocytes by using this transporter.     

Inhibition by anandamide of gap junctions and intercellular calcium signalling in striatal astrocytes

Anandamide, an endogenous arachidonic acid derivative that is released from neurons and activates cannabinoid receptors, may act as a transcellular cannabimimetic messenger in the central nervous system. The biological actions of anandamide and the identity of its target cells are, however, still poorly documented. Here we show that anandamide is a potent inhibitor of gap-junction conductance and dye permeability in striatal astrocytes. This inhibitory effect is specific for anandamide as compared to co-released congeners or structural analogues, is sensitive to pertussis toxin and to protein-alkylating agents, and is neither mimicked by cannabinoid-receptor agonists nor prevented by a cannabinoid-receptor antagonist. Glutamate released from neurons evokes calcium waves in astrocytes that propagate via gap junctions, and may, in turn, activate neurons distant from their initiation sites in astrocytes. We find that anandamide blocks the propagation of astrocyte calcium waves generated by either mechanical stimulation or local glutamate application. Thus, by regulating gap-junction permeability, anandamide may control intercellular communication in astrocytes and therefore neuron-glial interactions.     

Synthesis of secretory component by rat hepatocytes in culture

Normal rat hepatocytes were isolated and cultivated in vitro. Synthesis of secretory component was demonstrated by its accumulation in the culture medium, as measured by radioimmunoassay; by incorporation of 14C-leucine in the protein specifically precipitated with anti-secretory component antiserum; and by a positive precipitin reaction of concentrated culture medium with the same antiserum. The results explain the high levels of secretory component found in rat bile and render plausible a mechanism of hepatic IgA transfer involving secretory component as the hepatocyte membrane receptor for polymeric IgA.     

Inhibition by inorganic ions of a sustained calcium signal evoked by activation of mGlu5 receptors in rat cortical neurons and glia

The effect of mGlu receptor agonists on intracellular calcium (Ca2+) in rat cortical neurons and glial cells was studied. The responses evoked consisted of two phases; an initial transient response followed by a sustained plateau. In both cell types the order of potency of group I mGlu receptor agonists was DHPG > 1S,3R ACPD > 3-HPG. The selective mGlu5 agonist CHPG elicited responses in both cell types as did S4C3-HPG which is thought to be an mGlu5 agonist at high concentrations. S4-CPG had no effect on intracellular Ca2+ levels nor did it inhibit the action of IS,3R ACPD. These results suggest that the responses in both cell types are mediated by mGlu5 receptors. In the absence of extracellular Ca2+ ions, 1S,3R ACPD (100 microM) induced only a transient Ca2+ response which decayed to baseline with a time constant of approximately 20 s in both cell types. Subsequent readdition of Ca2+ (2 mM) to the external solution in the continued presence of 1S,3R ACPD induced a sustained Ca2+ plateau. The sustained Ca2+ plateau could be blocked by a number of inorganic cations, with an order of potency of Zn2+ > or = La3+ > Cd2+ > or = Co2+ > Ni2+ > Mg2+. Similar concentrations of Zn2+ had little effect on Ca2+-influx evoked by 25 mM K+. It is concluded that the Ca2+-entry pathway activated by mGlu5 receptors resembles store-operated Ca2+-entry pathways that have been described in other cell types.     

Arginine vasopressin triggers intracellular calcium release, a calcium-activated potassium current and exocytosis in identified rat corticotropes

Arginine vasopressin (AVP) stimulates the secretion of ACTH from pituitary corticotropes. We investigated the action of AVP in single corticotropes of male rats. Corticotropes were identified with the reverse hemolytic plaque assay using antibodies against ACTH. Using the whole-cell recording technique in conjunction with the fluorescent Ca2+ indicator, indo-1 to measure the concentration of cytosolic free Ca2+ ([Ca2+]i), we show that AVP triggers a transient and plateau pattern of Ca2+ signal. The [Ca2+]i elevation activates the apamin-sensitive Ca2+-activated K+ current, which, in turn, causes membrane hyperpolarization. The Ca2+ signal can be elicited in the absence of extracellular Ca2+ and is mimicked by intracellular inositol 1,4,5-trisphosphate (IP3). Both GDP-beta-S and heparin inhibit the AVP response. Thus, AVP triggers intracellular Ca2+ release from the (IP3)-sensitive store via a GTP binding protein-coupled phosphoinositide pathway. Using the high temporal resolution capacitance measurement to detect exocytosis in single corticotropes, we show that a burst of exocytosis is evoked during the AVP-triggered [Ca2+]i elevation. Exocytosis can also be triggered when Ca2+ is released directly from the IP3-sensitive store via flash photolysis of caged IP3. We conclude that AVP-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release from the IP3-sensitive store.     

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.     

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.     

Nuclear accumulation of G-actin in isolated rat hepatocytes by adenine nucleotides

Distinct inositol and phosphatidylinositol polyphosphates 5-phosphatases have recently been cloned. Primers have been designed coding for highly conserved amino acid regions that are shared between sequences of 5-phosphatases. One of the PCR fragment referred to as 51 C, shows 99% identity to a previously reported sequence (INPPL-1) present in the database. We report here the identification of cDNAs for a new SH2-domain-containing protein showing homology to the inositol 5-phosphatase SHIP and therefore referred to as SHIP2. SHIP2 differs at both N- and C-terminal ends with the sequence of INPPL-1. The translated sequence of SHIP2 encodes a 1258 amino acid protein with a predicted molecular mass of 142 kDa. Particularly high levels of SHIP2 were found in human heart, skeletal muscle and placenta as shown by Northern blot analysis. SHIP2 was also expressed in dog thyroid cells in primary culture where the expression was enhanced in TSH and EGF-stimulated cells.     

Atrial natriuretic peptide inhibits evoked catecholamine release by altering sensitivity to calcium

Natriuretic peptides are cyclized peptides produced by cardiovascular and neural tissues. These peptides inhibit various secretory responses such as the release of renin, aldosterone and autonomic neurotransmitters. This report tests the hypothesis that atrial natriuretic peptide reduces dopamine efflux from an adrenergic cell line, rat pheochromocytoma cells, by suppressing intracellular calcium concentrations. The L-type calcium channel inhibitor, nifedipine, markedly suppressed dopamine release from depolarized PC12 cells, suggesting that calcium entering through this channel was the predominant stimulus for dopamine efflux. Atrial natriuretic peptide maximally reduced depolarization-evoked dopamine release 20 +/- 3% at a concentration of 100 nM and this effect was abolished by nifedipine, but not by pretreatment with the N-type calcium channel inhibitor, omega-conotoxin, or an inhibitor of calcium-induced calcium release, ryanodine. In cells loaded with Fura-2, atrial natriuretic peptide both augmented depolarization-induced increases of intracellular free calcium concentrations and accelerated the depolarization-induced quenching of the Fura-2 signal by manganese, findings consistent with enhanced conductivity of calcium channels. Dopamine efflux induced by either the calcium ionophore, A23187, or staphylococcal alpha toxin was attenuated by atrial natriuretic peptide. Additionally, a natriuretic peptide interacting solely with the natriuretic peptide C receptor in these cells, C-type natriuretic peptide, also suppressed calcium-induced dopamine efflux in permeabilized cells. These data are consistent with natriuretic peptides attenuating catecholamine exocytosis in response to calcium but inconsistent with the neuromodulatory effect resulting from a reduction in intracellular calcium concentrations within pheochromocytoma cells.