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Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes in PM cation fluxes, cell death, and NLRP3 inflammasome activation., (Copyright © 2016 the American Physiological Society.)

Chronic activation of proinflammatory caspase-1 in the retinas of diabetic animals and patients in vivo and retinal Müller cells in vitro is well documented. In this study we characterized how elevated glucose and extracellular purines contribute to the activation of caspase-1 in a cultured rat Müller cell (rMC-1) model. The ability of high glucose (25 mM, 24 h) to activate caspase-1 was attenuated by either apyrase, which metabolizes extracellular ATP to AMP, or adenosine deaminase (ADA), which metabolizes extracellular adenosine to inosine. This suggested that autocrine stimulation of ATP-sensing P2 receptors and adenosine-sensing P1 receptors may in part mediate the response to high glucose. Exogenous ATP, 5'-N-ethylcarboxamido-adenosine (NECA), a nonselective P1 receptor agonist, or forskolin (FSK) increased caspase-1 activity in rMC-1 cells cultured in control glucose (5 mM) medium. Accumulation of active caspase-1 was also increased by dipyridamole, which suppresses adenosine reuptake. High-glucose stimulation of caspase-1 was attenuated by suramin, a nonselective P2 antagonist, or A2 adenosine receptor antagonists, but not by antagonism of P2X7 ATP-gated ion channel receptors. Although high glucose increased P2X7 mRNA, neither P2X7 protein nor function was detected in rMC-1 cells. The increased caspase-1 activity stimulated by high glucose, FSK, NECA, or ATP was correlated with increased gene expression of caspase-1 and thioredoxin-interacting-protein (TXNIP). These findings support a novel role for autocrine P1 and P2 purinergic receptors coupled to cAMP signaling cascades and transcriptional induction of caspase-1 in mediating the high-glucose-induced activation of caspase-1 and secretion of IL-1β in a cell culture model of nonhematopoietic retinal Müller cells.

Extracellular inorganic pyrophosphate (ePP(i)) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP(i) metabolism controls calcification. To determine the role of ePP(i) in vascular smooth muscle, we identified the pathways responsible for ePP(i) production and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1-3), the putative PP(i) transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP(i) from ATP in aortas was blocked by β,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1(-/-)) did not synthesize PP(i) from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP(i) could not be demonstrated in aortas, aortas from mutant (ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP(i) was reduced 25% by β,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP(i) was increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1(-/-) aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP(i), indicating an important inhibitory role of locally produced PP(i). Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP(i). NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.

Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PP(i)), and regulated generation and homeostasis of extracellular PP(i) is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PP(i) generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PP(i) mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (P(i)) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PP(i). Attenuated PP(i) accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PP(i). Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular P(i) alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated P(i) induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PP(i) accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) alpha-actin, SM22alpha, and calponin. Notably, addition of exogenous ATP (or PP(i) per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PP(i) generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification.

We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.

Extracellular inorganic pyrophosphate (PP(i)) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate PP(i) via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular PP(i) via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog beta,gamma-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular PP(i) to accumulate >or=600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed PP(i) accumulation while increasing ATP accumulation. Probenecid, which inhibits PP(i) efflux dependent on ANK, a putative PP(i) transporter or transport regulator, reduced extracellular PP(i) accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises >or=50% of the extracellular PP(i)-generating capacity of VSMC. The accumulation of extracellular PP(i) and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular PP(i) accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to PP(i) generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix.

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a G(q)/phophospholipase C (PLC)/Ca(2+) mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca(2+) mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain G(q)-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca(2+)-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.

The steady-state maintenance of highly asymmetric concentrations of the major inorganic cations and anions is a major function of both plasma membranes and the membranes of intracellular organelles. Homeostatic regulation of these ionic gradients is critical for most functions. Due to their charge, the movements of ions across biological membranes necessarily involves facilitation by intrinsic membrane transport proteins. The functional characterization and categorization of membrane transport proteins was a major focus of cell physiological research from the 1950s through the 1980s. On the basis of these functional analyses, ion transport proteins were broadly divided into two classes: channels and carrier-type transporters (which include exchangers, cotransporters, and ATP-driven ion pumps). Beginning in the mid-1980s, these functional analyses of ion transport and homeostasis were complemented by the cloning of genes encoding many ion channels and transporter proteins. Comparison of the predicted primary amino acid sequences and structures of functionally similar ion transport proteins facilitated their grouping within families and superfamilies of structurally related membrane proteins. Postgenomics research in ion transport biology increasingly involves two powerful approaches. One involves elucidation of the molecular structures, at the atomic level in some cases, of model ion transport proteins. The second uses the tools of cell biology to explore the cell-specific function or subcellular localization of ion transport proteins. This review will describe how these approaches have provided new, and sometimes surprising, insights regarding four major questions in current ion transporter research. 1) What are the fundamental differences between ion channels and ion transporters? 2) How does the interaction of an ion transport protein with so-called adapter proteins affect its subcellular localization or regulation by various intracellular signal transduction pathways? 3) How does the specific lipid composition of the local membrane microenvironment modulate the function of an ion transport protein? 4) How can the basic functional properties of a ubiquitously expressed ion transport protein vary depending on the cell type in which it is expressed?

The mechanisms underlying caspase-1 activation and IL-1beta processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1beta secretion. Analysis of in vitro IL-1beta and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1beta. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X(7) receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1beta. This acceleration of in vitro processing was strictly dependent on loss of intracellular K(+) from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K(+) loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca(2+)-independent phospholipase A(2). Caspase-1 activation and IL-1beta processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K(+) efflux via P2X(7) nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays.

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.

P2X receptors function as ATP-gated cation channels. The P2X(7) receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X(7) receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X(7) receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 microM ATP was sufficient for activation of nonselective pores by P2X(7) receptors. Comparison of P2X(7) receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X(7) receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X(7) receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X(7) receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.

The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X(7) ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X(7) receptors or 2) MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-gamma, a maneuver known to upregulate P2Z/P2X(7) receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X(7) receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X(7) channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)); the initial increase reflects MTX-induced Ca(2+) influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca(2+)](i) and ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X(7) receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X(7) receptor, MTX-induced increases in [Ca(2+)](i) and ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca(2+)](i) had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22 degrees C, a characteristic shared by the P2Z/P2X(7)-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X(7) receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

We have developed a method for measuring the local concentration of ATP at the extracellular surface of live cells. This method relies on the specific attachment to the cell surface of a chimeric protein that consists of the IgG-binding domain of Staphylococcus aureus protein A fused in-frame with the complete sequence for firefly luciferase (proA-luc). Expression of proA-luc in Escherichia coli and its one-step affinity purification are straightforward. Attachment to cells is demonstrated to be specific and antibody dependent using several suspended and adherent cell types. Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, and sufficient to provide nanomolar sensitivity. The spatial resolution of this method enables the observation of strictly local changes in extracellular ATP during its secretion from activated platelets. Furthermore, the activity of cell-attached luciferase is relatively refractory to the inclusion of nucleotidases in the medium, arguing for its effectiveness in cell systems possessing potent ecto-ATPases.

The expression of P2 purinergic receptor subtypes in leukocytes varies with both lineage and developmental stage. Given the recent identification and cloning of at least seven distinct G protein-coupled ATP receptor subtypes (P2Y family), we investigated P2Y receptor subtype expression during myeloid cell differentiation. We observed that KG-1 myeloblasts express P2Y1 but not P2Y2 receptors (previously termed P2U receptors), whereas later myeloid progenitors, including HL-60 promyelocytes and THP-1 monocytes, expressed P2Y2 but not P2Y1 receptors. In KG-1 cells, significant activation of Ca2+ mobilization by P2Y1 receptors was only observed after preincubation with potato apyrase, an exogenous ATPase. This indicated that P2Y1 receptors are desensitized in KG-1 cells by autocrine mechanisms that may involve enhanced release of endogenous nucleotides and/or decreased expression of cell-surface ecto-nucleotidases. We compared the levels of ecto-apyrase activity and expression in KG-1 myeloblasts and HL-60 promyelocytes. Extracellular ATP was rapidly metabolized by HL-60 but not by KG-1 cells. Reverse transcription-polymerase chain reaction analysis indicated that mRNA for CD39 (cluster of differentiation), an identified ecto-apyrase, was present in HL-60 but not KG-1 cells. Ecto-apyrase activity was modestly increased with differentiation of myeloid progenitors with either phorbol 12-myristate 13-acetate (PMA) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). Differentiation of HL-60 cells with PMA, but not DBcAMP, strongly induced ecto-5'-nucleotidase activity and CD73 mRNA expression. These observations indicate that signal transduction by extracellular ATP in myeloid leukocytes can be regulated by developmentally programmed changes in the expression of P2Y receptor subtypes and multiple ecto-nucleotidases.

Vascular smooth muscle cells (VSMC) contribute to the pathophysiology of hypertension through cell growth and contraction, and phospholipase C (PLC) is a critical effector enzyme in growth factor and vasoconstrictor signaling. There is indirect evidence that angiotensin II (ANG II) receptors are linked to the PLC-beta isoform signaling pathways. However, recent studies suggest that PLC-beta isoforms may not be expressed in VSMC. Our data demonstrate that in human aortic VSMC, PLC-beta 1 and PLC-gamma 1 proteins were detected by immunoblot analysis, and PLC-beta 1 mRNA was identified by reverse transcriptase-polymerase chain reaction in rat aortic VSMC. Incubation of permeabilized VSMC with anti-PLC-beta 1 or anti-Gq alpha antibodies inhibited ANG II-dependent inositol polyphosphate (IP) formation, while anti-PLC-gamma 1 antibodies did not inhibit ANG II-regulated IP formation. Conversely, anti-PLC-gamma 1 antibodies completely abolished platelet-derived growth factor (PDGF)-dependent IP generation, whereas anti-PLC-beta 1 antibodies had no effect on PDGF-induced PLC activation. Inhibition of tyrosine phosphorylation with genistein or herbimycin A did not diminish ANG II-stimulated IP formation or cytosolic free Ca2+ concentration transients, thereby confirming that ANG II signals via a PLC-gamma 1-independent mechanism. In summary, PLC-beta 1 and PLC-gamma 1 are expressed in human aortic VSMC, and PLC-beta 1 is the isoform that is critical for ANG II-regulated PLC signaling in these cells.

Vasoconstrictor hormones contribute to the pathogenesis of hypertension through intracellular signals that stimulate vascular smooth muscle (VSMC) contraction and/or growth. We previously showed that the glucocorticoid dexamethasone (DEX) inhibited angiotensin II-stimulated inositol trisphosphate (IP3) formation in VSMC, but the mechanism of inhibition is not known. Because glucocorticoids stimulate the expression of annexins and annexin II potently binds phosphoinositides, the role of DEX and annexin II in VSMC G protein-coupled phosphoinositide hydrolysis was investigated. DEX incubation blunted increases in guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-stimulated IP3 generation and angiotensin II-induced intracellular Ca2+ mobilization but stimulated elevations in VSMC annexin II content. VSMC incubation with exogenous purified annexin II resulted in concentration-dependent decreases in GTP gamma S-stimulated IP3 formation. In DEX-treated cells, exogenous annexin II did not further diminish GTP gamma S-stimulated IP3 formation, suggesting that endogenous annexin II may be a mediator of DEX-induced inhibition of G protein-coupled IP3 generation. These data represent the first direct evidence of G protein-dependent phosphoinositide hydrolysis regulation by glucocorticoids or annexins. We speculate that annexin II may play a role in the pathogenesis of hypertension through stimulation of VSMC growth.

Exposure of Clone 9 cells (a rat liver cell line expressing only the GLUT-1 isoform) to 5 mM azide or to 3 microM ionomycin for 12 h results in 3.7 +/- 0.3- and 4.9 +/- 0.4-fold increases in GLUT-1 mRNA content, respectively, suggesting the hypothesis that a rise in cytosolic free calcium concentration ([Ca2+]i) mediates the induction of GLUT-1 mRNA by azide. Five lines of evidence were employed to test this hypothesis. 1) Exposure of cells to 0-3 microM of ionomycin increased [Ca2+]i from 83 +/- 9 to 504 +/- 20 nM (half-maximal effect at 0.1 microM ionomycin), whereas half-maximal increase in GLUT-1 mRNA occurred at 1 microM ionomycin, with the increase in the mRNA being negligible at [Ca2+]i below 400 nM. Exposure of cells to 5 mM azide, however, increased [Ca2+]i to maximal value of 174 +/- 22 nM at 15 s, suggesting that the magnitude of the increase in [Ca2+]i by azide may not be adequate for the response. 2) The increase in GLUT-1 mRNA content by azide was fully preserved in cells preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 3) GLUT-1 mRNA content increased within 30 min of exposure to ionomycin, whereas the mRNA increased after a "delay" period of 2 h in cells exposed to 5 mM azide. 4) A brief (2-min) rise in [Ca2+]i by ionomycin was sufficient to increase GLUT-1 mRNA content, whereas continuous exposure to azide for > 1 h was necessary for a subsequent induction of the mRNA. 5) Treatment with ionomycin, A-23187, and thapsigargin caused larger increases in glucose-regulated protein 78 and 94 and in 70-kDa heat shock protein mRNAs than in GLUT-1 mRNA, whereas treatment with azide resulted in greater induction of GLUT-1 mRNA. These results strongly suggest that, whereas increased [Ca2+]i enhances GLUT-1 mRNA expression and azide increases [Ca2+]i, the rise in [Ca2+]i does not mediate the induction of GLUT-1 mRNA in response to inhibition of oxidative phosphorylation.

We tested the hypothesis that an increase in cytosolic calcium concentration stimulates glucose transporter isoform (GLUT-1) gene expression. Exposure of a rat liver cell line (Clone 9) to 3 microM A-23187 for 12 h resulted in 3-, 5-, and 10-fold increases in cytochalasin B-inhibitable 3-O-methyl-D-glucose transport, GLUT-1 protein, and GLUT-1 mRNA content, respectively. The induction of GLUT-1 mRNA in response to A-23187 is not preceded by a significant decrease in cell ATP content. This induction is prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in conjunction with ethylene glycol-bis(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid. To investigate the mechanism of GLUT-1 mRNA induction, we found that exposure to A-23187 stabilized GLUT-1 mRNA: with the employment of actinomycin D, GLUT-1 mRNA had a half-life of 1.5 and 5.5 h in control and A-23187-treated cells, respectively. In nuclear run-on assays, the rate of GLUT-1 gene transcription was stimulated 1.5- to 1.7-fold in nuclei isolated from cells exposed to A-23187 for either 30 min or 2 h. These results demonstrate that exposure to A-23187 stimulates GLUT-1 gene expression and that the increase in GLUT-1 mRNA content is mediated in part by enhanced GLUT-1 gene transcription as well as decreased GLUT-1 mRNA degradation. The increase in GLUT-1 mRNA content, in turn, is associated with increased cell GLUT-1 content and enhanced glucose transport.

Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.

Platelet-derived growth factor (PDGF), a key mitogen for liver fat-storing cells (FSC), is a dimeric molecule that occurs as homodimers or heterodimers of related polypeptide chains (PDGF-BB, -AB, and -AA). In chronic inflammation of the liver lobule, any of the three dimeric forms of PDGF derived from multiple sources could potentially interact with FSC. We explored the effects of the three different PDGF isoforms on DNA synthesis and early signal transduction pathways potentially related to PDGF mitogenicity in rat liver FSC. PDGF-BB homodimer and -AB heterodimer induced a marked increase in DNA synthesis, whereas the effect of PDGF-AA homodimer was considerably lower. Moreover, the mitogenicity of each isoform proportionally correlated with their effects on phosphoinositide turnover and intracellular Ca2+. Both the PDGF-BB and -AB dimers likely interact with the PDGF-beta-receptor, although PDGF-AB requires at least one alpha-receptor. The low responsiveness to PDGF-AA could not be accounted for by downregulation of the PDGF-alpha-receptor because FSC expressed very low levels of PDGF-A- and B-chain mRNAs and did not secrete detectable amounts of PDGF activity in the conditioned media. In addition, preincubation of FSC with suramin, a potent inhibitor of PDGF binding to its receptor, failed to increase PDGF-AA-induced DNA synthesis. These results are consistent with a predominant expression of PDGF-beta-receptor in liver FSC, that is linked to phospholipase C activation.

Prostaglandins (PG) and thromboxane A2 (TxA2) have marked vasoactive effects on the renal glomerular microcirculation. Exposure of cultured mesangial cells to PGF2 alpha and TxA2 mimetics results in a rapid elevation of free cytosolic Ca2+ ([Ca2+]i) followed by contraction and cell proliferation. We studied whether other ionic changes mediate these effects of eicosanoids on cells of rat and human origin. Cytoplasmic pH (pHi) was monitored in cells loaded with the fluorescent, intracellularly trapped pH-sensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. PGF2 alpha in rat cells and the TxA2 mimetic U-46619 in human cells induced rapid, dose-dependent cytosolic acidification followed by recovery and net alkalinization mediated by enhanced Na(+)-H+ exchange. The early acidification was also stimulated by ionomycin and Ca2(+)-mobilizing peptides, implicating a Ca2(+)-dependent mechanism. Alkalinization was abolished by removal of extracellular Na+ and by amiloride. Both components of the responses were inhibited by phorbol myristate acetate, which could mimic alkalinization, suggesting a regulatory role of protein kinase C in activation of the Na(+)-H+ exchanger by eicosanoids. Vasoconstrictor arachidonate metabolites may control glomerular cell function by a signaling mechanism centered on concurrent changes of pHi and [Ca2+]i.

Platelet-derived growth factor (PDGF) is a potent mitogen and vasoactive polypeptide for aortic smooth muscle. Because contractile glomerular mesangial cells synthesize a PDGF-like molecule and may respond to PDGF released by infiltrating cells at the site of glomerular inflammation, we studied the effects of exogenous, highly purified PDGF on 1) contraction of cultured rat mesangial cells and 2) membrane phosphoinositide turnover and cytosolic free calcium ([Ca2+]i), as putative mechanisms of membrane signal transduction. PDGF, 10(-11) and 10(-10) M, contracted 56.1 +/- 5.2 and 72.9 +/- 6.4% of the cells, respectively, with an average decrease of cross-sectional area of 22.0 +/- 2.6 and 28.1 +/- 2.7% of basal, as assessed by image-analysis microscopy. PDGF also rapidly increased total water-soluble inositol phosphates, measured after anion-exchange chromatography on perchloric acid-extracted cells, and simultaneously raised [Ca2+]i, measured by the fluorescent intracellular probe fura-2, from basal levels of 83.1 +/- 6.8 to a peak of 229.4 +/- 20.0 nM. We conclude that PDGF stimulates contraction of rat mesangial cells via a phospholipase C-dependent pathway, with potential relevance to the control of glomerular hemodynamics and mesangial proliferation in immune-mediated glomerular disease.

Although peptidoleukotriene (LTC4, LTD4) receptors have been characterized by radioligand binding studies, pathways of transmembrane signaling by activated leukotriene receptors remain obscure. We employed [3H]LTD4 binding studies and fluorescent measurements of intracellular Ca2+ concentration ([Ca2+]) and pH to identify LTD4 receptors and mechanisms of transmembrane signaling in cultured human mesangial cells. Mesangial cells expressed a single class of saturable, specific binding sites for [3H]LTD4. Kinetic, competition, and saturation analyses gave an average KD of approximately 12.0 nM with a Bmax of 987 fmol/mg protein. LTC4 competed with high affinity for [3H]LTD4 binding sites, as did LTB4 but with much lower affinity. [3H]LTD4 binding was blocked by a specific LTD4 receptor antagonist, SKF 102922. LTD4 and LTC4 also evoked a rapid (2-3 s), transient increase in intracellular [Ca2+], followed by a second, sustained increase. The transient phase was independent of extracellular Ca2+, whereas the sustained phase was dependent on extracellular Ca2+. Intracellular [Ca2+] was unaffected by LTB4. The LTD4-stimulated Ca2+ transients were dose dependent (1 nM-1 microM) and, similar to [3H]LTD4 binding, Ca2+ transients were inhibited by LTD4 receptor antagonists. We also report evidence that LTD4 affects intracellular pH and activates Na+-H+ exchange. Specifically, LTD4 induced an initial acidification within 1-2 min, followed by net alkalinization at 5 min. Alkalinization was due to activation of an amiloride-inhibitable Na+-H+ exchanger. LTD4 receptors were apparently not coupled to adenylate cyclase or phospholipase A2 as we detected no changes of adenosine 3',5'-cyclic monophosphate (cAMP) or prostanoids. Thus we conclude that [3H]LTD4 binding sites on human mesangial cells are coupled to a Ca2+-signaling system and Na+-H+ exchange. Moreover LTD4, a potent inflammatory mediator, failed to stimulate cAMP or prostaglandin E2/prostaglandin I2, two counterregulatory autacoids that preserve normal mesangial function.

Phospholipase C activation by prostaglandins (PG) and thromboxane A2 (TxA2) was studied in cultured rat and human glomerular mesangial cells, measuring accumulation of radiolabeled inositol phosphates and cytosolic free calcium ([Ca2+]i) with the fluorescent intracellular probe fura-2. Prostaglandin F2 alpha (PGF2 alpha) and TxA2 were found to be the major eicosanoids active on this signaling pathway in rat and human cells, respectively, whereas other PG had lesser or no effects. PGF2 alpha and TxA2 rapidly induced accumulation of inositol trisphosphate accompanied by a simultaneous transient rise of [Ca2+]i, followed by sustained elevation or, in human cells, by a distinct second increase of [Ca2+]i within 45 s. A minor initial accumulation of inositol monophosphate was followed by marked elevation greater than 5 min after the early responses. Responses to different eicosanoids were mediated by separate receptors, functionally characterized using receptor antagonists or heterologous desensitization during sequential applications. Protein kinase C activation by serum and phorbol esters potently inhibited inositol phosphate accumulation and/or [Ca2+]i transients, indicating a pathway for a negative feedback on PG-evoked intracellular signals. We conclude that receptor-mediated phospholipase C activation underlies the biological effects of certain eicosanoids on the glomerular mesangium.

Teased renal tubules of the winter flounder (Pseudopleuronectes americanus) were employed to investigate the structural requirements for two pathways of D-glucose transport which take place preponderantly across the basal (antiluminal) face of renal cells. 1) An inhibition analysis of the equilibrating, Na-independent and phlorizin-sensitive transport of the nonmetabolizable methyl-alpha-D-glucoside (0.1 and 0.5 mM), with 20 glucose analogs (5 mM), was employed to establish the structural requirements for the substrate-carrier interaction: a (pyranose) ring, oxygen, or F at C1, C2-OH, C3-OH, and C4-OH (all axial, 1C model). Some interaction may also occur at C6-OH. D-Glucose shares this transport system. Hydrogen bonding between the oxygens and the carrier is suggested. 2) The phloretin- and phlorizin-sensitive, ouabain-insensitive transport of D-glucose, 2-deoxy-D-glucose, and D-mannose is associated with considerable phosphorylation. The three sugars mutually compete for a shared transport site. The specificity pattern characterizing the transport system defines the following structural requirements: a (pyranose) ring, a free C1-OH, C3-OH, and C4-OH (both axial) and possibly C6-OH. Hydrogen bonding between the carrier and the oxygens at C3, C4, and C6, and covalent bonding at C1 is suggested.

Glomerulonephritis is frequently associated with immunoglobulin deposition in the mesangium. We had previously shown that contractile, rat mesangial cells in culture synthesize superoxide anion after binding immune complexes (IC) in a manner dependent on the Fc region of immunoglobulin G (IgG). We now studied the effects of soluble IC on mesangial cell cytosolic free calcium ([Ca2+]i) and phosphatidylinositol turnover as putative mechanisms of transmembrane signaling as well as prostaglandin biosynthesis and contraction. IC (500 micrograms specific antibody) raised [Ca2+]i in mesangial cells loaded with fura-2 from resting levels of 100.4 +/- 8.0 to a peak of 282.3 +/- 31.5 nM in a dose-dependent manner. Removal of extracellular Ca2+ by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid only slightly reduced peak, IC-stimulated [Ca2+]i to 236 +/- 18 nM but prevented the sustained phase of the response, indicating that IC both mobilized Ca2+ from intracellular stores and increased the influx of Ca2+ across the plasma membrane. IC did not increase water-soluble inositol phosphates, measured by anion-exchange chromatography of trichloroacetic acid-extracted cells but markedly stimulated PGE2 and thromboxane B2 synthesis in a dose- and time-dependent manner. Finally, IC (250 micrograms specific antibody) induced 45.8 +/- 10.1% of the cells to contract with an average decrease in cross-sectional surface area of 20.0 +/- 1.8% of basal as assessed by image-analysis microscopy. IC formed with F(ab')2 fragments of antibody and antigen or mixtures of antigen and nonimmune whole molecule antibody did not alter [Ca2+]i, induce prostaglandin synthesis, or stimulate mesangial cell contraction.(ABSTRACT TRUNCATED AT 250 WORDS)