Sphingosine-1-phosphate induces airway smooth muscle hyperresponsiveness and proliferation.

BACKGROUND: The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could represent a potential therapeutic target in asthma and other lung diseases. Several studies in animal surrogates have suggested a role for S1P-mediated signaling in the regulation of airway smooth muscle (ASM) contraction, airway hyperresponsiveness, and airway remodeling, but evidence from human studies is lacking. OBJECTIVE: We sought to compare the responsiveness of the airways to S1P in healthy and asthmatic individuals in vivo, in isolated human airways ex vivo, and in murine airways dissected from healthy and house dust mite (HDM)-sensitized animals. METHODS: Airway responsiveness was measured by spirometry during inhalation challenges and by wire myography in airways isolated from human and mouse lungs. Thymidine incorporation and calcium mobilization assays were used to study human ASM cell responses. RESULTS: S1P did not induce contraction of airways isolated from healthy and HDM-exposed mice, nor in human airways. Similarly, there was no airway constriction observed in healthy and asthmatic subjects in response to increasing concentrations of inhaled S1P. However, a 30-minute exposure to S1P induced a significant concentration-dependent enhancement of airway reactivity to methacholine and to histamine in murine and human airways, respectively. HDM-sensitized mice demonstrated a significant increase in methacholine responsiveness, which was not further enhanced by S1P treatment. S1P also concentration-dependently enhanced proliferation of human ASM cells, an effect mediated through S1P receptor type 2, as shown by selective antagonism and S1P receptor type 2 small-interfering RNA knockdown. CONCLUSIONS: Our data suggest that S1P released locally into the airways may be involved in the regulation of ASM hyperresponsiveness and hyperplasia, defining a novel target for future therapies.

Role of capacitative Ca2+ entry but not Na+/Ca2+ exchange in hypoxic pulmonary vasoconstriction in rat intrapulmonary arteries.

The identities of the Ca2+ influx mechanisms underlying hypoxic pulmonary vasoconstriction (HPV), the contractile response of pulmonary arteries (PA) to hypoxia, remain controversial. We investigated the roles of Na+/Ca2+ exchange (NCX) and capacitative Ca2+ entry (CCE) in HPV by measuring isometric tension in rat intrapulmonary arteries (IPA) during hypoxia. It has been shown in PA cells that hypoxia raised [Ca2+]i by inhibiting NCX. We found that removal of Na+ caused a response resembling HPV, suggesting that HPV could be due to inhibition of NCX. However, prior inhibition of NCX using Na+ free solution or 3 microM KB-R7943 enhanced rather than prevented HPV, indicating that HPV is not caused by an inhibition of NCX. The CCE blocker 2-APB inhibited both phases of HPV, and the ryanodine receptor blocker dantrolene inhibited the 2nd phase of HPV. Taken together with previous observations, these results suggest a role for CCE in HPV, but suggest that different CCE pathways may be involved for each phase.

Capacitative calcium entry as a pulmonary specific vasoconstrictor mechanism in small muscular arteries of the rat.

(1) The effect of induction of capacitative Ca2+ entry (CCE) upon tone in small (i.d. 200-500 microm) intrapulmonary (IPA), mesenteric (MA), renal (RA), femoral (FA), and coronary arteries (CA) of the rat was examined. (2) Following incubation of IPA with 100 nm thapsigargin (Thg) in Ca2+-free physiological salt solution (PSS), a sustained contraction was observed upon reintroduction of 1.8 mm Ca2+, which was unaffected by either diltiazem (10 microm) or the reverse mode Na+/Ca2+ antiport inhibitor KB-R7943 (10 microm). An identical protocol failed to elicit contraction in MA, RA, or CA, while a small transient contraction was sometimes observed in FA. (3) The effect of this protocol on the intracellular Ca2+ concentration ([Ca2+]i) was assessed using Fura PE3-loaded IPA, MA, and FA. Reintroduction of Ca2+ into the bath solution following Thg treatment in Ca2+-free PSS caused a large, rapid, and sustained increase in [Ca2+]i in all the three types of artery. (4) 100 nm Thg induced a slowly developing noisy inward current in smooth muscle cells (SMC) isolated from IPA, which was due to an increase in the activity of single channels with a conductance of approximately 30 pS. The current had a reversal potential near 0 mV in normal PSS, and persisted when Ca2+-dependent K+ and Cl- currents were blocked; it was greatly inhibited by 1 microm La3+, 1 microm Gd3+, and the IP3 receptor antagonist 2-APB (75 microm), and by replacement of extracellular cations by NMDG+. (5) In conclusion, depletion of intracellular Ca2+ stores with Thg caused capacitative Ca2+ entry in rat small muscular IPA, MA, and FA. However, a corresponding contraction was observed only in IPA. CCE in IPA was associated with the development of a small La3+- and Gd3+-sensitive current, and an increased Mn2+ quench of Fura PE-3 fluorescence. These results suggest that although CCE occurs in a number of types of small arteries, its coupling to contraction appears to be of particular importance in pulmonary arteries.

Hypoxic pulmonary vasoconstriction: mechanisms and controversies.

The pulmonary circulation differs from the systemic in several important aspects, the most important being that pulmonary arteries constrict to moderate physiological (20-60 mmHg PO2) hypoxia, whereas systemic arteries vasodilate. This phenomenon is called hypoxic pulmonary vasoconstriction (HPV), and is responsible for maintaining the ventilation-perfusion ratio during localized alveolar hypoxia. In disease, however, global hypoxia results in a detrimental increase in total pulmonary vascular resistance, and increased load on the right heart. Despite many years of study, the precise mechanisms underlying HPV remain unresolved. However, as we argue below, there is now overwhelming evidence that hypoxia can stimulate several pathways leading to a rise in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMC). This rise in [Ca2+]i is consistently found to be relatively small, and HPV seems also to require rho kinase-mediated Ca2+ sensitization. There is good evidence that HPV also has an as yet unexplained endothelium dependency. In this brief review, we highlight selected recent findings and ongoing controversies which continue to animate the study of this remarkable and unique response of the pulmonary vasculature to hypoxia.

Mechanisms of the prostaglandin F2alpha-induced rise in [Ca2+]i in rat intrapulmonary arteries.

The mechanisms by which prostaglandin F(2alpha) (PGF(2alpha)) increases intracellular Ca2+ concentration [Ca2+]i in vascular smooth muscle remain unclear. We examined the role of store-, receptor- and voltage-operated Ca2+ influx pathways in rat intrapulmonary arteries (IPA) loaded with Fura PE-3. Low concentrations (0.01-1 microM) of PGF(2alpha) caused a transient followed by a plateau rise in [Ca2+]i. Both responses became maximal at 0.1 microM PGF(2alpha). At higher concentrations of PGF(2alpha), a further slower rise in [Ca2+]i was superimposed on the plateau. The [Ca2+]i response to 0.1 microM PGF(2alpha) was mimicked by the FP receptor agonist fluprostenol, whilst the effect of 10 microM PGF(2alpha) was mimicked by the TP receptor agonist U-46619. The plateau rise in [Ca2+]i in response to 0.1 microM PGF(2alpha) was insensitive to diltiazem, and was abolished in Ca2+-free physiological salt solution, and by pretreatment with La3+, 2-APB, thapsigargin or U-73122. The rises in [Ca2+]i in response to 10 microM PGF(2alpha) and 0.01 microM U-46619 were partially inhibited by diltiazem. The diltiazem-resistant components of both of these responses were inhibited by 2-APB and La3+ to an extent which was significantly less than that seen for the response to 0.1 microM PGF(2alpha), and were also much less sensitive to U-73122. The U-46619 response was also relatively insensitive to thapsigargin. When Ca2+ was replaced with Sr2+, the sustained increase in the Fura PE-3 signal to 0.1 microM PGF(2alpha) was abolished, whereas 10 microM PGF(2alpha) and 0.05 microM U-46619 still caused substantial increases. These results suggest that low concentrations of PGF(2alpha) act via FP receptors to cause IP3-dependent Ca2+ release and store operated Ca2+ entry (SOCE). U-46619 and 10-100 microM PGF(2alpha) cause a TP receptor-mediated Ca2+ influx involving both L-type Ca2+ channels and a receptor operated pathway, which differs from SOCE in its susceptibility to La3+, 2-APB and thapsigargin, does not require phospholipase C activation, and is Sr2+ permeable.