Genetic counselling for pulmonary arterial hypertension: a matter of variable variability.

We report three cases which highlight the complex considerations surrounding genetic counselling for pulmonary arterial hypertension (PAH). The first counselee developed PAH symptoms shortly after his daughter's death from PAH and was diagnosed with a delay of 1 year. An early diagnosis of familial PAH was established in the second counselee. Oral therapy was initiated immediately, and her functional status has since remained stable. The third counselee was a healthy woman who struggled to cope with her risk for familial PAH, having lost two siblings from the disease. These cases show that incomplete penetrance and variable expression need particular attention during clinical assessment and genetic counselling of heritable PAH patients and family members.

Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells.

Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.

Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell-cell communication.

Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK(+)) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK(+) and, to a lesser extent, with wild-type c-Src, but not with kinase-dead c-Src. Mutation of residue Cx43 Tyr(265) (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK(+) in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43Delta263) that lacks residue Tyr(265). Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr(265), resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.

Malignant effusions contain lysophosphatidic acid (LPA)-like activity.

BACKGROUND: Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids with mitogenic and growth factor-like activities that act via specific cell-surface receptors present in many normal and transformed cell types. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. The presence of LPA-like activity and the hypothesis that levels of this bioactivity in effusions of ovarian cancer patients are higher than those in effusions of other cancer patients was studied. MATERIALS AND METHODS: A neurite retraction bioassay in a neuroblastoma cell line previously developed for in vitro detection of LPA activity on cell lines was employed and bioactivity was expressed in virtual LPA-equivalent levels. LPA-equivalent levels were tested in effusions of 62 patients with a range of malignancies, including 13 ovarian cancer patients. Biochemical and clinical parameters were evaluated for correlations with LPA-equivalent levels. RESULTS: Average LPA-equivalent levels were 50.2 microns (range 5.4-200) for all patients, and 94.5 microns (range 15-200) for ovarian cancer patients (P = 0.004). There were no additional independent significant correlations between LPA-equivalent levels in effusions and a range of other biochemical and clinical characteristics. CONCLUSION: These data suggest a role for LPA-like lipids in the peritoneal spread of ovarian cancer and possibly that of other predominantly intraperitoneal malignancies.

Exogenous phospholipase D generates lysophosphatidic acid and activates Ras, Rho and Ca2+ signaling pathways.

BACKGROUND: Phospholipase D (PLD) hydrolyzes phospholipids to generate phosphatidic acid (PA) and a free headgroup. PLDs occur as both intracellular and secreted forms; the latter can act as potent virulence factors. Exogenous PLD has growth-factor-like properties, in that it induces proto-oncogene transcription, mitogenesis and cytoskeletal changes in target cells. The underlying mechanism is unknown, although it is generally assumed that PLD action is mediated by PA serving as a putative second messenger. RESULTS: In quiescent fibroblasts, exogenous PLD (from Streptomyces chromofuscus) stimulated accumulation of the GTP-bound form of Ras, activation of mitogen-activated protein (MAP) kinase and DNA synthesis, through the pertussis-toxin-sensitive inhibitory G protein Gi. Furthermore, PLD mimicked bioactive lysophospholipids (but not PA) in inducing Ca2+ mobilization, membrane depolarization and Rho-mediated neurite retraction. PLD action was mediated by Iysophosphatidic acid (LPA) derived from Iysophosphatidylcholine acting on cognate G-protein-coupled LPA receptor(s). There was no evidence for the involvement of PA in mediating the effects of exogenous PLD. CONCLUSIONS: Our results provide a molecular explanation for the multiple cellular responses to exogenous PLDs. These PLDs generate bioactive LPA from pre-existing Iysophosphatidylcholine in the outer membrane leaflet, resulting in activation of G-protein-coupled LPA receptors and consequent activation of Ras, Rho and Ca2+ signaling pathways. Unscheduled activation of LPA receptors may underlie, at least in part, the known pathogenic effects of exogenous PLDs.

Acute loss of cell-cell communication caused by G protein-coupled receptors: a critical role for c-Src.

Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.

Sphingosine 1-phosphate signalling through the G-protein-coupled receptor Edg-1.

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are structurally related lipid mediators that act on distinct G-protein-coupled receptors to evoke similar responses, including Ca2+ mobilization, adenylate cyclase inhibition, and mitogen-activated protein (MAP) kinase activation. However, little is still known about the respective receptors. A recently cloned putative LPA receptor (Vzg-1/Edg-2) is similar to an orphan Gi-coupled receptor termed Edg-1. Here we show that expression of Edg-1 in Sf9 and COS-7 cells results in inhibition of adenylate cyclase and activation of MAP kinase (Gi-mediated), but not Ca2+ mobilization, in response to S1P. These responses are specific in that (i) S1P action is not mimicked by LPA, and (ii) Vzg-1/Edg-2 cannot substitute for Edg-1. Thus the Edg-1 receptor is capable of mediating a subset of the cellular responses to S1P.

Lysophosphatidic acid: G-protein signalling and cellular responses.

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates a specific G protein coupled receptor to evoke multiple cellular responses. Recent work has identified two cDNAs encoding putative LPA receptors, various LPA-like agonists that act on distinct receptors, and new pathways that link the receptor(s) to such diverse events as Ras signalling, cytoskeletal remodelling and membrane depolarization.

Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein-coupled receptors to mediate similar actions.

Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor.

Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membrane depolarization by activating a new type of Cl- channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers.

Lysophosphatidic acid-induced Ca2+ mobilization in human A431 cells: structure-activity analysis.

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells, which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered, the rank order of potency is oleoyl-LPA > arachidonoyl-LPA > linolenoyl-LPA > linoleoyl-LPA > stearoyl-LPA = palmitoyl-LPA > myristoyl-LPA. The shorter-chain species, lauroyl- and decanoyl-LPA, show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses > 15 microM. Similarly, the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate, a putative messenger with some structural similarities to LPA, elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however, cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that, although many features of the LPA structure are important for optimal activity, the phosphate group is most critical, suggesting that this moiety is directly involved in receptor activation.

Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action.

Lysophosphatidic acid (LPA) is a mitogenic phospholipid produced by certain activated cells and present in serum. LPA stimulates phospholipase C and inhibits adenylate cyclase in its target cells, apparently by activating a specific G-protein-coupled receptor. Here, we demonstrate that LPA causes transient rounding of N1E-115 and NG108-15 neuronal cells accompanied by growth cone collapse and retraction of neurites. The effect of LPA is concentration dependent, being half-maximal at 10-20 nM, and reversibly blocked by suramin, an LPA receptor antagonist. The morphological response to LPA is indistinguishable from that evoked by thrombin or a thrombin receptor-activating peptide (TRP) (K. Jalink and W. H. Moolenaar, J. Cell Biol., 118: 411-419, 1992); yet, LPA and thrombin appear to act through distinct receptors. LPA-induced shape changes, like those induced by thrombin and TRP, are driven by contraction of the cortical actin cytoskeleton and not attributable to prior phospholipid hydrolysis and Ca2+ mobilization nor to other classic second messenger systems. Instead, LPA- and TRP-induced shape changes are accompanied by a small but significant increase in p60src protein tyrosine kinase activity. Treatment of cells with pervanadate selectively inhibits LPA- and TRP-induced shape changes as well as p60src activation. These results indicate that, in N1E-115 and NG108-15 cells, LPA and TRP trigger neurite retraction and cell rounding through a novel, receptor-mediated signaling pathway, and they suggest that p60src may play a role in this pathway.