Participation of the microtubular-microfilamentous system on intracellular Ca2+ transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[14C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca2+. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca2+, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca2+ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca2+. In addition, an increase in cytosolic Ca2+ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca2+, suggesting that Ca2+ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca2+ increase induced by A23187 in a Ca2+-free medium. On the other hand, in a Ca2+-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca2+ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca2+ uptake. These observations indicate that intracellular Ca2+ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca2+ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Direct demonstration of increases in cytosolic free Ca2+ during stimulation of pancreatic enzyme secretion

The Ca²⁺-activated photoprotein aequorin has been incorporated into intact, isolated rat pancreatic acini by a hypotonic swelling method. The isolated acini retained normal secretory responses after loading with aequorin. Increases in cytosolic Ca²⁺ concentration in response to a physiological secretagogue, carbamylcholine, and to divalent-cation ionophore A23187 have been demonstrated. Simultaneous measurement of the dynamics of enzyme secretion and changes in cytosolic Ca²⁺ concentration has been achieved using a newly developed apparatus.     

The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells

The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca²⁺]i, in bovine adrenal chromaffin cells was determined. At 10^–4M carbamylcholine maximal release occurred with an accompanying increase i n [Ca²⁺]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca²⁺]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca²⁺]i.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca²⁺ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca²⁺ as well as PG synthesis. The increase of cytosolic Ca²⁺ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca²⁺ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGIA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca²⁺, whereas TMB-8 (10 μM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca²⁺, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca²⁺.These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca²⁺ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca²⁺ and on the other hand, PG synthesis.     

Secretion of granule enzymes from alveolar macrophages : Regulation by intracellular Ca-buffering capacity

Rabbit alveolar macrophages exposed to the ionophore A23187 in the presence of extracellular Ca²⁺ take up about 12 nmoles of Ca²⁺/1 × 10⁶ cells. This uptake induces a slight, but significant, extracellular release of granule enzymes, β-glucuronidase and lysozyme, but not of the cytoplasmic marker, lactate dehydrogenase. If either EGTA is added to the medium or Mg²⁺ replaces Ca²⁺ no stimulation of secretion is observed. If the energy supply is decreased by treating the macrophages with mitochondrial inhibitors such as oligomycin, cyanide, or rotenone and antimycin, Ca²⁺-dependent secretion is potentiated several fold. Selective release of granule enzymes from macrophages exposed to A23187 and Ca²⁺ is also stimulated by cytochalasin B (CB).     

Translational suppression by Ca2+ ionophores: Reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca²⁺ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50–300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca²⁺ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca²⁺ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH₃ pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca²⁺ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca²⁺. Ionomycin produced more Ca²⁺ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca²⁺ by ionomycin were readily reversed in GH₃ cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca²⁺ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca²⁺ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca²⁺-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca²⁺ accumulation. Increased Ca²⁺ contents in response to Ca²⁺ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.Abbreviations BSA bovine serum albumin - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - PKR double-stranded RNA-regulated protein kinase - ER endoplasmic reticulum - eIF eukaryotic initiation factor     

Effect of protein kinase C activation and Ca2+ mobilization on hexose transport in Swiss 3T3 cells

Down-modulation of Ca²⁺-activated, phospholipid-dependent protein kinase (protein binase C), which was accomplished by pretreatment with phorbol-12,13-dibutyrate for 24 h, resulted in the loss of a phorbol ester-induced stimulation of hexose transport activity in Swiss 3T3 cells. In these cells, however, platelet-derived growth factor as well as Ca²⁺ ionophore A23187 were still able to induce stimulation of hexose transport activity accompanied by the elevation of intracellular free Ca²⁺ concentration. Since chelation of extracellular Ca²⁺ inhibited this stimulation, inflow of extracellular Ca²⁺ into cytoplasm seemed to be esential for the stimulatory effect of platelet-derived growth factor and A23187 on hexose transport. Epidermal growth factor and insulin also stimulated hexose transport activity regardless of the absence of protein kinase C. However, in the case of epidermal growth factor, intracellular Ca²⁺, but not extracellular Ca²⁺, was found to be necessary for the stimulation. On the other hand, insulin stimulated the hexose transport independent of both intra- and extracellular Ca²⁺.     

Relation between cytoskeleton,hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells

Summary Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca²⁺ or Ca²⁺-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca²⁺ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K⁺ ionophore valinomycin.     

The peripheral Ca2+ pump activity of macrophages and neutrophils

Exposure of either alveolar macrophages or blood neutrophils to 0.2 – 1 μM ionophore A23187 in the presence of 0.1 – 1 mM CaCl₂ causes a rapid extracellular release of Ca²⁺, which can be measured by a Ca²⁺-selective electrode. The initial rate at which the cation is extruded from the cells is about 0.1 – 0.2 μg-ions/min/ml of cell water. ATP depletion, but not replacement of extracellular Na⁺ with choline, produces a marked inhibition of Ca²⁺ release from macrophages. When the movements of Ca²⁺ between neutrophils and the incubation medium are followed by an isotopic technique, a transient increase in cell-associated ⁴⁵Ca²⁺ is detected a few seconds after the addition of the ionophore. We suggest that the ionophore A23187 mobilises Ca²⁺ from intracellular stores, with a subsequent cell extrusion of the bivalent cation catalysed by a pump localised at the cell surface. These and other data are consistent with the conclusion that the peripheral Ca²⁺ pump system of macrophages and neutrophils is very similar to the well know Ca²⁺ pump of the red cells with regard to mechanism and capacity.     

Intracellular uptake and α-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells

Summary Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca²⁺ and Mg²⁺ (t1/2=1 min) and much slower in the presence of Ca²⁺ or Mg²⁺ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca²⁺ and Mg²⁺, and by equivalent interaction of ionophore with membranes of whole and sonicated cells.A23187 (10 m) increased amylase release 200% in the presence of extracellular Ca²⁺ and Mg²⁺. In the absence of Ca²⁺ (but in the presence of Mg²⁺) A23187 did not increase amylase release. A23187 (10 m) also produced Ca²⁺-dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 m) increased amylase release 140% without increasing LDH release or permeability to trypan blue.     

Calcium requirements in the action of cholecystokinin-pancreozymin on pancreatic acinar cell metabolism.

The present study utilized ionophore A23187 to determine the role of Ca²⁺ in pancreatic acinar cell metabolism. The ionophore A23187 in the presence of EGTA increased efflux of Ca²⁺ from the rat pancreatic fragments. Ionophore and CCK-PZ were equally effective in the presence of extracellular Ca²⁺ in stimulating ¹⁴C-labeled protein secretion. The ionophore decreased synthesis of new protein more effectively than CCK-PZ in the presence of extracellular Ca²⁺. The effect of ionophore and CCK-PZ in combination was greater than either agent alone. Phospholipid labeling was not stimulated by A23187 in the presence of extracellular Ca²⁺ in contrast to CCK-PZ. With CCK-PZ, the effect was dependent on the concentration of extracellular Ca²⁺. Protein phosphorylation was stimulated ～ 109% by CCK-PZ and ～ 39% by ionophore. CCK-PZ stimulated protein phosphorylation in the 100,000 g supernatant whereas A23187 was ineffective. Ionophore A23187 inhibited glucose oxidation whereas CCK-PZ stimulated glucose oxidation. These data suggest that more than one kinase system might be involved in metabolic responses to hormonal stimulation of the pancreas viz. a phosphorylase kinase may be directly activated by Ca²⁺ causing protein discharge whereas other kinase system may require binding of the hormone to receptor leading to other events besides protein discharge.     

Calcium ionophore-induced changes in HCO3− secretion and Cl− absorption in turtle bladder: relation to action of 3-isobutyl-1-methylxanthine

The calcium ionophore A23187 stimulates luminal alkalinization and inhibits Cl⁻ absorption in short-circuited urinary bladders of postprandial or alkalotic turtles. The ionophore appears to mimic the action of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) by its similar effects on HCO₃⁻ secretion and Cl⁻ absorption and by increasing cytosolic cAMP levels of isolated bladder epithelial cells. However, only A23187 (or ionomycin), but not IMBX or cAMP, elevated cytosolic Ca²⁺ of aequorin- or quin2-loaded cells. Since A23187, but not IBMX or cAMP inhibits luminal acidification, we postulate that cytosolic Ca²⁺ (1) regulates the acidification process by a cAMP-independent mechanism and (2) controls HCO₃⁻ secretion as well as Cl⁻ absorption, at least in part, via cAMP-mediated pathways.     

Calcium-dependent K efflux from rat submandibular gland: The effects of trifluoperazine and quinidine

The Ca²⁺-dependent K⁺ efflux from rat submandibular gland was studied using a K⁺-sensitive electrode. A K⁺ efflux was induced by either adrenalin or by using the divalent cation ionophore A23187 plus added Ca²⁺ to bypass the receptor mechanism. Trifluoperazine, which was used to investigate the role of calmodulin, was found to block the adrenalin-induced K⁺ efflux but not the A23187/Ca²⁺-induced K⁺ efflux. The adrenalin-induced K⁺ efflux was abolished by quinidine and the A23187/Ca²⁺-induced K⁺ efflux was significantly reduced by quinidine. In other experiments, the presence of indomethacin did not inhibit the adrenalin-induced K⁺ efflux, and exogenously added arachidonic acid did not induce a K⁺ efflux. It is concluded that neither prostaglandin synthesis, nor a cytosolic Ca²⁺-calmodulin complex is involved in the agonist-induced K⁺ efflux from rat submandibular gland. A similarity between the Ca²⁺-dependent K⁺ efflux mechanism of erythrocyte ghosts and submandibular tissue is indicated by their common response to quinidine.     

Calmodulin-dependent and -independent apoptosis in cells of a Drosophila neuronal cell line

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca²⁺ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca²⁺ ions, although the origin of Ca²⁺ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca²⁺ ions whereas those by the other three chemicals utilized intracellular Ca²⁺ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).     

Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

Effects of Ca ionophore A23187 and Ca deficiency on pancreatic phospholipids and amylase release in vitro

The Ca²⁺ ionophore A23187 elicits a transient increase in pancreatic amylase release in vitro, and this is accompanied by a transient decrease in phosphatidyl inositol concentration. Effects of ionophore A23187 and carbachol on amylase release and phosphatidylinositol breakdown are dependent on medium Ca²⁺. These results suggest that major secretagogue-induced, pancreatic phospholipid changes follow, rather than precede, changes in Ca²⁺ in the pancreas.     

Tentacle contraction inDiscophrya collini: The effects of ionophore A23187 and ruthenium red on Ca2+-induced contraction and uptake of extracellular calcium

Summary The tentacles of the suctorian protozoonDiscophrya collini are stimulated to contract by externally applied Ca²⁺. The role of extracellular Ca²⁺ in tentacle contraction was studied by monitoring⁴⁵Ca²⁺ uptake, using ionophore A23187 to facilitate membrane transport of calcium and ruthenium red (RR) as an inhibitor of transport. The degree of tentacle retraction was dependent upon external Ca²⁺ concentration and studies with⁴⁵Ca²⁺ using scintillation counting indicated a linear relationship between external Ca²⁺ concentration and Ca²⁺ uptake. Uptake of Ca²⁺ was enhanced in the presence of the ionophore while RR caused little inhibition.⁴⁵Ca²⁺ uptake was only partially inhibited by RR when cells were subjected to a Ca²⁺, ionophore and RR mixture. Grain counts from light microscope autoradiographs after treatment of cells with⁴⁵Ca²⁺/ionophore,⁴⁵Ca²⁺/RR or⁴⁵Ca²⁺ alone showed heavy, light and intermediate labelling respectively. In all instances the grains were evenly distributed within the cell.These observations are interpreted as supporting the suggestion that the ionophore enhances both the uptake of extracellular Ca²⁺ and release of Ca²⁺from an internal source, while the RR could only partially prevent movement of Ca²⁺ through the plasma mebrane. A model is presented suggesting that tentacle retraction is mediated by cytosolic Ca²⁺ levels which are determined by the fluxing of Ca²⁺ across the plasma membrane and the membrane of elongate dense bodies which act as internal Ca²⁺ reservoirs.     

Relationship Between the Actions of Calcium Ions, Opioids and Prostaglandin E1 on the Level of Cyclic AMP in Neuroblastoma × Glioma Hybrid Cells

Abstract: Neuroblastoma × glioma hybrid cells increase their intracellular concentration of cyclic AMP in response to prostaglandin E₁ (PGE₁). This effect is inhibited by opioids. The response to PGE₁ is positively correlated with the concentration of Ca²⁺ in the incubation medium. The Ca²⁺ antagonists Co²⁺ and La³⁺, the Ca²⁺ chelator EGTA and a blocker of Ca²⁺ influx into cells, Segontin, inhibit the response to PGE₁. At low external concentrations of Ca²⁺ the response to PGE₁ is enhanced by the Ca²⁺ ionophore A23187. The effects of A23187 and Segontin point to a cytosolic site of Ca²⁺ action. Lack of Ca²⁺ reduces the level of cyclic AMP even in the absence of PGE₁ and the presence of an inhibitor of cyclic AMP phosphodiesterase. Ca²⁺ is required even for an increase in the level of cyclic AMP in cells pretreated with cholera toxin. The increases in level of cyclic AMP evoked by PGE, in a neuroblastoma and by PGE₁ or noradrenaline in a glioma cell line do not depend on Ca²⁺. The response of the hybrid cells to the opioid leucine-enkephalin appears not to rely on the presence of Ca²⁺. Even changing the intracellular concentration of Ca²⁺ by the ionophore A23187 does not alter the effect of the opioid. The analogy between opioids and lack of Ca²⁺ in the short-term (minutes) experiments mentioned holds also for long-term (hours) experiments. Cells chronically exposed to opioids or to low concentrations of Ca²⁺ display an enhanced maximal response to PGE₁.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Role of transglutaminase 1 in stabilisation of intercellular junctions of the vascular endothelium

Microvascular endothelial monolayers from mouse myocardium (MyEnd) cultured for up to 5 days postconfluency became increasingly resistant to various barrier-compromising stimuli such as low extracellular Ca²⁺ and treatment with the Ca²⁺ ionophore A23187 and with the actin depolymerising compound cytochalasin D. In contrast, microvascular endothelial monolayers from mouse lung microvessels (PulmEnd) remained sensitive to these conditions during the entire culture period which corresponds to the well-known in vivo sensitivity of the lung microvasculature to Ca²⁺ depletion and cytochalasin D treatment. One molecular difference between pulmonary and myocardial endothelial cells was found to be transglutaminase 1 (TGase1) which is strongly expressed in myocardial endothelial cells but is absent from pulmonary endothelial cells. Resistance of MyEnd cells to barrier-breaking conditions correlated strongly with translocation of TGase1 to intercellular junctions. Simultaneous inhibition of intracellular and extracellular TGase activity by monodansylcadaverine (MDC) strongly weakened barrier properties of MyEnd monolayers, whereas inhibition of extracellular TGases by the membrane-impermeable active site-directed TGase inhibitor R281 did not reduce barrier properties. Weakening of barrier properties could be also induced in MyEnd cells by downregulation of TGase1 expression using RNAi-based gene silencing. These findings suggest that crosslinking activity of intracellular TGase1 at intercellular junctions may play a role in controlling barrier properties of endothelial monolayers.