Growth of Endothelial Cells in Space and in Simulated Microgravity - a Comparison on the Secretory Level.

BACKGROUND/AIMS: Endothelial cells exposed to the Random Positioning Machine (RPM) reveal three different phenotypes. They grow as a two-dimensional monolayer and form three-dimensional (3D) structures such as spheroids and tubular constructs. As part of the ESA-SPHEROIDS project we want to understand how endothelial cells (ECs) react and adapt to long-term microgravity. METHODS: During a spaceflight to the International Space Station (ISS) and a subsequent stay onboard, human ECs (EA.hy926 cell line) were cultured for 12 days in real microgravity inside an automatic flight hardware, specially designed for use in space. ECs were cultivated in the absence or presence of vascular endothelial growth factor, which had demonstrated a cell-protective effect on ECs exposed to an RPM simulating microgravity. After cell fixation in space and return of the samples, we examined cell morphology and analyzed supernatants by Multianalyte Profiling technology. RESULTS: The fixed samples comprised 3D multicellular spheroids and tube-like structures in addition to monolayer cells, which are exclusively observed during growth under Earth gravity (1g). Within the 3D aggregates we detected enhanced collagen and laminin. The supernatant analysis unveiled alterations in secretion of several growth factors, cytokines, and extracellular matrix components as compared to cells cultivated at 1g or on the RPM. This confirmed an influence of gravity on interacting key proteins and genes and demonstrated a flight hardware impact on the endothelial secretome. CONCLUSION: Since formation of tube-like aggregates was observed only on the RPM and during spaceflight, we conclude that microgravity may be the major cause for ECs' 3D aggregation.

Directed self-assembly of spheroids into modular vascular beds for engineering large tissue constructs.

Spheroids can be used as building-blocks for bottom-up generation of artificial vascular beds, but current biofabrication strategies are often time-consuming and complex. Also, pre-optimization of single spheroid properties is often neglected. Here, we report a simple setup for rapid biomanufacturing of spheroid-based patch-like vascular beds. Prior to patch assembly, spheroids combining mesenchymal stem/stromal cells (MSCs) and outgrowth endothelial cells (OECs) at different ratios (10:1; 5:1; 1:1; 1:5) were formed in non-adhesive microwells and monitored along 7 d. Optimal OEC retention and organization was observed at 1:1 MSC/OEC ratio. Dynamic remodelling of spheroids led to changes in both cellular and extracellular matrix components (ECMs) over time. Some OEC formed internal clusters, while others organized into a peripheral monolayer, stabilized by ECM and pericyte-like cells, with concomitant increase in surface stiffness. Along spheroid culture, OEC switched from an active to a quiescent state, and their endothelial sprouting potential was significantly abrogated, suggesting that immature spheroids may be more therapeutically relevant. Non-adhesive moulds were subsequently used for triggering rapid, one-step, spheroid formation/fusion into square-shaped patches, with spheroids uniformly interspaced via a thin cell layer. The high surface area, endothelial sprouting potential, and scalability of the developed spheroid-based patches make them stand out as artificial vascular beds for modular engineering of large tissue constructs.

Engineering injectable vascularized tissues from the bottom-up: Dynamics of in-gel extra-spheroid dermal tissue assembly.

Modular tissue engineering approaches open up exciting perspectives for the biofabrication of vascularized tissues from the bottom-up, using micro-sized units such as spheroids as building blocks. While several techniques for 3D spheroid formation from multiple cell types have been reported, strategies to elicit the extra-spheroid assembly of complex vascularized tissues are still scarce. Here we describe an injectable approach to generate vascularized dermal tissue, as an example application, from spheroids combining fibroblasts and endothelial progenitors (OEC) in a xeno-free (XF) setting. Short-term cultured spheroids (1 day) were selected over mature spheroids (7 days), as they showed significantly higher angiogenic sprouting potential. Embedding spheroids in fibrin was crucial for triggering cell migration into the external milieu, while providing a 3D framework for in-gel extra-spheroid morphogenesis. Migrating fibroblasts proliferated and produced endogenous ECM forming a dense tissue, while OEC self-assembled into stable capillaries with lumen and basal lamina. Massive in vitro interconnection between sprouts from neighbouring spheroids rapidly settled an intricate vascular plexus. Upon injection into the chorioallantoic membrane of chick embryos, fibrin-entrapped pre-vascularized XF spheroids developed into a macrotissue with evident host vessel infiltration. After only 4 days, perfused chimeric capillaries with human cells were present in proximal areas, showing fast and functional inosculation between host and donor vessels. This method for generating dense vascularized tissue from injectable building blocks is clinically relevant and potentially useful for a range of applications.

Antiphosphotyrosine recovery of phospholipase C activity after EGF treatment of A-431 cells.

A tenfold increase in phospholipase C activity specific for phosphatidylinositol 4,5-bisphosphate (PIP2) was immunopurified from extracts of A-431 epidermoid carcinoma cells stimulated with epidermal growth factor. This finding suggests a biochemical link between growth factor-stimulated tyrosine kinase activity and PIP2 hydrolysis.

Continuous Exposure to Simulated Hypergravity-Induced Changes in Proliferation, Morphology, and Gene Expression of Human Tendon Cells.

Gravity influences physical and biological processes, especially during development and homeostasis of several tissues in the human body. Studies under altered gravity have been receiving great attention toward a better understanding of microgravity-, hypogravity (<1 g)-, or hypergravity (>1 g)-induced alterations. In this work, the influence of simulated hypergravity over human tendon-derived cells (hTDCs) was studied at 5, 10, 15, and 20 g for 4 or 16 h, using a large diameter centrifuge. Main results showed that 16 h of simulated hypergravity limited cell proliferation. Cell area was higher in hTDCs cultured at 5, 10, and 15 g for 16 h, in comparison to 1 g control. Actin filaments were more pronounced in hTDCs cultured at 5 and 10 g for 16 h. Focal adhesion kinase (FAK) was mainly expressed in focal adhesion sites upon hypergravity stimulation, in comparison to perinuclear localization in control cells after 16 h; and FAK number/cell increased with increasing g-levels. A tendency toward an upregulation of tenogenic markers was observed; scleraxis (SCX), tenascin C (TNC), collagen type III (COL3A1), and decorin (DCN) were significantly upregulated in hTDCs cultured at 15 g and COL3A1 and DCN were significantly upregulated in hTDCs cultured at 20 g. Overall, simulated hypergravity affected the behavior of hTDCs, with more pronounced effects in the long-term period (16 h) of stimulation.

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis.

Cyclooxygenase-2 (COX-2) inhibitors reduce angiogenic responses to a variety of stimuli, suggesting that products of COX-2 may mediate critical steps. Here, we show that thromboxane A2 (TXA2) is one of several eicosanoid products generated by activated human microvascular endothelial cells. Selective COX-2 antagonists inhibit TXA2 production, endothelial migration, and fibroblast growth factor-induced corneal angiogenesis. Endothelial migration and corneal angiogenesis are similarly inhibited by a TXA2 receptor antagonist, SQ29548. A TXA2 agonist, U46619, reconstitutes both migration and angiogenesis responses under COX-2-inhibited conditions. These findings identify TXA2 as a COX-2 product that functions as a critical intermediary of angiogenesis.

Effects of hypergravity on the angiogenic potential of endothelial cells.

Angiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothelial behaviour in altered gravity might contribute to new insights towards angiogenesis modulation. This study aimed at characterizing EC behaviour after hypergravity exposure (more than 1g), with special focus on cytoskeleton architecture and capillary-like structure formation. Herein, human umbilical vein ECs (HUVECs) were cultured under two-dimensional and three-dimensional conditions at 3g and 10g for 4 and 16 h inside the large diameter centrifuge at the European Space Research and Technology Centre (ESTEC) of the European Space Agency. Although no significant tendency regarding cytoskeleton organization was observed for cells exposed to high g's, a slight loss of the perinuclear localization of ß-tubulin was observed for cells exposed to 3g with less pronounced peripheral bodies of actin when compared with 1g control cells. Additionally, hypergravity exposure decreased the assembly of HUVECs into capillary-like structures, with a 10g level significantly reducing their organization capacity. In conclusion, short-term hypergravity seems to affect EC phenotype and their angiogenic potential in a time and g-level-dependent manner.

5' untranslated sequences determine degradative pathway for alternate PDGF B/c-sis mRNA's.

The 5' untranslated sequence (5' UTS) of the platelet-derived growth factor B (PDGF B/c-sis) mRNA is GC rich, includes AUGs upstream of the translation initiation site, and inhibits translation of downstream heterologous and autologous coding sequences. Its primary sequence has been remarkably conserved through evolution. In this study, we identified two additional PDGF B/c-sis mRNAs expressed in endothelial cells. These were shown by Northern, primer extension, and nuclease protection analyses to differ in extent of 5' untranslated first exon sequence. The predominant of these 5' truncated transcripts was 2.8kb, extended 15nt upstream of the translation start site and was markedly stabilized by cycloheximide and anisomycin, but not puromycin or pactamycin. Cycloheximide increased the half-life of this mRNA approximately 7 fold, without affecting stability of the full length mRNA. Cycloheximide withdrawal caused its abrupt destabilization. The 2.8kb PDGF B/c-sis mRNA lacks translation inhibitory 5' UTS, encodes the same PDGF B protein, appears initiated from within first exon genomic sequences, and is degraded through a process that is sensitive to translation arrest and distinct from that degrading the 3.8kb PDGF B/c-sis mRNA.

Eph family receptors and ligands in vascular cell targeting and assembly.

Members of the Eph family of receptor tyrosine kinases determine neural cell aggregation and targeting behavior, functions that are also critical in vascular assembly and remodeling. Among this class of diverse receptors, EphA2 (Eck) and EphB1 (ELK) represent prototypes for two receptor subfamilies distinguished by high-affinity interaction with either glycerophosphatidylinositol (GPI)-linked or transmembrane ligands, respectively. EphA2 participates in angiogenic responses to tumor necrosis factor (TNF) through an autocrine loop affecting endothelial cell migration. EphB1 and its ligand Ephrin-B1 (LERK-2) are important determinants of assembly of endothelial cells from the microvasculature of the kidney, where both are expressed in endothelial progenitors and in glomerular microvascular endothelial cells. Ephrin-B1 activation of EphB1 promotes assembly of these cells into capillary-like structures. Interaction trap approaches have identified downstream signaling proteins that complex with ligand-activated EphA2 or EphB1, including nonreceptor tyrosine kinases and SH2 domain-containing adapter proteins. The Grb 10 adapter is one of a subset that binds activated EphB1, but not EphA2, defining distinct signaling mechanisms for these related endothelial receptors. On the basis of observations in vascular endothelial cells and recent results defining Eph receptor and ligand roles in neural cell targeting, we propose that these receptors direct cell-cell recognition events that are critical in vasculogenesis and angiogenesis. (Trends Cardiovasc Med 1997;7:329-334). © 1997, Elsevier Science Inc.

Rate of CRL4(CRBN) substrate Ikaros and Aiolos degradation underlies differential activity of lenalidomide and pomalidomide in multiple myeloma cells by regulation of c-Myc and IRF4.

Recent discoveries suggest that the critical events leading to the anti-proliferative activity of the IMiD immunomodulatory agents lenalidomide and pomalidomide in multiple myeloma (MM) cells are initiated by Cereblon-dependent ubiquitination and proteasomal degradation of substrate proteins Ikaros (IKZF1) and Aiolos (IKZF3). By performing kinetic analyses, we found that the downregulation or proteasomal degradation of Ikaros and Aiolos led to specific and sequential downregulation of c-Myc followed by IRF4 and subsequent growth inhibition and apoptosis. Notably, to ensure growth inhibition and cell death, sustained downregulation of Ikaros and Aiolos, c-Myc or IRF4 expression was required. In addition, we found that the half-maximal rate, rather than the final extent of Ikaros and Aiolos degradation, correlated to the relative efficacy of growth inhibition by lenalidomide or pomalidomide. Finally, we observed that all four transcription factors were elevated in primary MM samples compared with normal plasma cells. Taken together, our results suggest a functional link between Ikaros and Aiolos, and the pathological dysregulation of c-Myc and IRF4, and provide a new mechanistic understanding of the relative efficacy of lenalidomide and pomalidomide based on the kinetics of substrate degradation and downregulation of their downstream targets.

Platelet-derived growth factor in renal development and disease.

In aggregate, the evidence reviewed here supports a very important role for PDGF expression and action at local glomerular and interstitial sites in human kidney development and disease. PDGF delivered by platelets, or produced by endogenous cells of the kidney is capable of stimulating responses including proliferation, matrix deposition, chemotaxis, and contraction in renal cells, particularly mesangial cells and interstitial fibroblasts. During kidney development, PDGF may mediate processes of cellular recruitment, extracellular matrix deposition, and proliferation with the constructive outcome of glomerulogenesis and vascularization. Proliferation and production of extracellular matrix components by these cells likely contribute to destructive proliferative and sclerotic responses attending proliferative and other glomerulopathies. As additional information accumulates, therapeutic targets within the PDGF system may provide opportunities to arrest destructive renal processes.

Origins and formation of microvasculature in the developing kidney.

Regulation of microvessel assembly in the developing kidney is not known and may occur through vasculogenic, angiogenic, or both processes. To examine this question, we grafted rat and mice embryonic (E) day 12 (E12) kidneys, which have only a rudimentary vasculature, into anterior eye chambers of mouse and rat hosts. Species-specific, monoclonal anti-basement membrane antibodies showed that glomerular basement membranes, mesangial matrices, and microvessel basement membranes were always derived from the graft. When wild-type E12 mouse kidneys were grafted into anterior chambers of ROSA26 mice, in which the beta-galactosidase transgene is expressed ubiquitously, glomerular and microvascular endothelial cells stemmed from the graft, even after maintenance of kidneys in organ culture for 6 days before grafting. Immunolabeling with antibodies against the vascular endothelial growth factor (VEGF) receptor, Flk1, the EphB1 receptor, and its ligand, ephrin-B1, labeled discrete mesenchymal cells in embryonic and newborn kidney cortex, as well as developing microvessel and glomerular endothelium. In adult kidneys, Flk1 labeled glomeruli weakly, other vascular structures were unlabeled. When wild-type E12 kidneys were grafted under renal capsules of adult ROSA26 hosts, endothelium developing within the graft again came from the implanted kidney. In contrast, when E12 kidneys were grafted into renal cortices of newborns, glomeruli within grafts now contained host-derived endothelium. Similarly, when ROSA26 E12 kidneys were implanted into newborn wild-type hosts, chimeric vessels containing graft- and host-derived endothelium were seen in nearby host tissue. Our results indicate that cells capable of forming the entire microvascular tree of grafted metanephroi are already present in the E12 kidney. We hypothesize that Flk1/VEGF and EphB1/ephrin-B1 mediate renal endothelial mitosis-motility and cell guidance-aggregation behavior, respectively.

ELK and LERK-2 in developing kidney and microvascular endothelial assembly.

Eph family receptor tyrosine kinases direct neuronal cell targeting, bundling and intercellular aggregation activity, yet their role in mammalian kidney development has been unexplored to date. We recently identified expression of ELK (Eph-like kinase) receptors in cultured human renal microvascular endothelial cells (HRMEC), and showed that ELK mediates their in vitro assembly into capillary-like structures in response to the exogenous ligand, LERK-2. Here we identify expression of the ELK ligand, LERK-2, in HRMEC and in primitive vascular structures of developing murine kidney. ELK and LERK-2 are expressed on endothelial progenitor cells of primitive microvasculature in a pattern similar to that of the VEGF receptor, flk-1. ELK LERK-2 and flk-1 antigens are also displayed on the branching ureteric bud epithelium; ELK and LERK-2 expression persists in mature collecting ducts, glomeruli and arterioles. To explore whether renal-derived endothelial cells may distinguish LERK-2 from the angiogenic Eck ligand, LERK-1 (B61), and whether endothelial cells from different sources may distinguish among Eph receptor ligands, we compared HRMEC and human umbilical vein endothelial cell (HUVEC) responses in an in vitro capillary-like assembly assay. HRMEC endothelial cells assembled capillary-like structures in response to LERK-2, but not LERK-1, under conditions that promoted HUVEC to assemble in response to LERK-1, but not LERK-2. Therefore, responses mediated through specific Eph family receptors (ELK and Eck) are discriminated by endothelial cells from different vascular bed sources. ELK and its ligand, LERK-2, are spatially and temporally coordinated in expression and may function in morphogenesis of the renal microvasculature.

High-efficiency and low-toxicity adenovirus-assisted endothelial transfection.

Cultured endothelial cells have provided a powerful tool for discovery of the molecular regulators of a range of vascular processes from angiogenesis to fibrinolysis (1). Yet, the utility of genetic manipulation of endothelial culture systems to dissect critical intracellular signaling processes has been limited to date. Available methods such as retroviral transduction require endothelial proliferation, while cationic lipid mediated transfection is inefficient and evokes marked toxicity in cultured endothelial cells (2-4). Adenoviral transduction of endothelial cells is efficient, but preparation of recombinant adenovirus vectors is cumbersome.

Identification of a subpopulation of human renal microvascular endothelial cells with capacity to form capillary-like cord and tube structures.

Endothelial specialization is a prominent feature within distinct capillary beds of organs such as mammalian kidney, yet immunological markers for functionally distinct subpopulations of cultured endothelial cells from tissue sources such as kidney have not been available. We developed a simple and reproducible isolation and culture procedure to recover human renal microvascular endothelial cells (HRMEC) from the cortex of unused donor kidneys. This procedure yields highly purified preparations of cells that display endothelial markers that include Factor VIII antigen, acetyl-LDL receptors, and determinants that bind Ulex europaeus lectin. HRMEC assemble into capillary-like cord and tube structures when plated on the surface of basement membrane-like matrix (BMM) in media containing phorbol myristate acetate. To further define subpopulations of HRMEC, we generated a panel of monoclonal antibodies and screened for those recognizing cell surface determinants. One monoclonal antibody recovery from this screen recognized a cell surface protein expressed on a subpopulation of HRMEC that we have designated PEC-1 (pioneer endothelial cell antigen-1). Cells expressing PEC-1 extended long, interconnecting filopodial processes in response to phorbol myristate acetate and assembled into capillary-like structures when plated on BMM. Anti-PEC-1 immunoprecipitated proteins of 25 and 27 kDa. Magnetic bead separation of PEC-1 (+) cells selected cells that assemble into capillary-like cord and tube structures. The remaining PEC-1 (-) HRMEC population formed matrix adherent patches. In the kidney, the PEC-1 determinant is expressed on a small subpopulation of microvascular glomerular cells and is prominently expressed on the apical membrane of proximal tubule cells. The PEC-1 determinant discriminates among subpopulations of HRMEC, identifying a subpopulation that contributes to assembly of capillary-like structures.

Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide.

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN-DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN(YW/AA). Overexpression of CRBN wild-type protein, but not CRBN(YW/AA) mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21(WAF-1) expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Distinct pathways mediate transcriptional regulation of platelet-derived growth factor B/c-sis expression.

The biochemical mechanisms responsible for regulating cellular platelet-derived growth factor expression are incompletely understood. Our previous studies have shown that platelet-derived growth factor B/c-sis mRNA levels are induced in human renal microvascular endothelial cells by either thrombin or transforming growth factor (TGF-beta), while exposure to agents which elevate cAMP levels blocks the induction responses. The current studies use combined transcription run-off and message decay rate experiments to show greater than 3-fold increases in rate of transcription after stimulation with either thrombin or TGF-beta. c-sis message has a 70-90-min half-life under basal conditions that is effectively unaltered by thrombin or TGF-beta. Forskolin does not decrease the stability of c-sis mRNA, although it attenuates transcription increases seen with inducing agents. TGF-beta induction of c-sis transcription is mediated independent of the protein kinase C (Ca2+- and phospholipid-dependent enzyme)-mediated responses to phorbol ester, as it remains intact following down-regulation of protein kinase C response; TGF-beta and phorbol elicit additive induction. Inhibitory effects of cAMP upon transcription act distal to early thrombin-receptor-coupled increases in phosphatidylinositol turnover and are capable of turning off TGF-beta-activated transcription after activation has been established. Both inducing and suppressing agents alter endothelial platelet-derived growth factor B/c-sis mRNA expression dominantly through effects upon rates of transcription, cAMP suppression of transcription is dominant, and TGF-beta and phorbol esters mediate induction of transcription through distinct pathways.

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

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

Cyclosporin A inhibits kinase C-independent activation of the Na+/H+ exchanger by PDGF and vanadate.

Mitogen-induced activation of Na+/H+ exchange was studied in Swiss 3T3 fibroblasts. Phorbol myristic acetate (PMA) caused amiloride inhibitable cell alkalinization. PDGF and vanadate, but not bombesin or thrombin, caused additional alkalinization when given 10 min after a maximal dose of PMA. Down-regulation of kinase C by 24 hr PMA exposure prevented the alkalinization response to bombesin and thrombin, but not to PDGF or vanadate. Cyclosporin A specifically blocked the additional alkalinization after PDGF or vanadate in cells acutely exposed to PMA and in kinase C down-regulated cells. Thus, there are at least two independent pathways which activate Na+/H+ exchange. PMA, bombesin, and thrombin act via kinase C. PDGF and vanadate cause additional stimulation of the Na+/H+ exchanger by a kinase C-independent pathway, inhibitable by cyclosporin A.