Gregory Knock

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.

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

The effect of induction of capacitative Ca2+ entry (CCE) upon tone in small (i.d. 200–500 μm) intrapulmonary (IPA), mesenteric (MA), renal (RA), femoral (FA), and coronary arteries (CA) of the rat was examined. 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 μM) or the reverse mode Na+/Ca2+ antiport inhibitor KB‐R7943 (10 μM). An identical protocol failed to elicit contraction in MA, RA, or CA, while a small transient contraction was sometimes observed in FA. 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. 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 ∼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 μM La3+, 1 μM Gd3+, and the IP3 receptor antagonist 2‐APB (75 μM), and by replacement of extracellular cations by NMDG+. 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.

Voltage‐gated K+ currents in freshly isolated myocytes of the pregnant human myometrium

1 Voltage‐gated K+ currents in human myometrium are not well characterized, and were therefore investigated, using the whole‐cell patch clamp technique, in freshly isolated myometrial smooth muscle cells from pregnant women at term. 2 Three types of voltage‐gated K+ currents were identified. IK1 was a 4‐aminopyridine‐insensitive current with a negative half‐inactivation (V0.5= ‐61 to ‐67 mV) and negative activation characteristics (threshold between ‐60 and ‐40 mV) and slow kinetics. IK2 was a 4‐aminopyridine‐sensitive current (half‐maximal block at ≈1 mM) with relatively positive half‐inactivation (V0.5= ‐30 mV) and activation characteristics (threshold between ‐40 and ‐30 mV) and faster kinetics. IK,A was a 4‐aminopyridine‐sensitive current with a negative inactivation and very fast inactivation kinetics. 3 Both IK1 and IK2 were sensitive to high concentrations of tetraethylammonium (half‐maximal block at ≈3 mM) and low concentrations of clofilium (half‐maximal block by 3‐10 μM). 4 I K1 and IK2 were unevenly distributed between myometrial cells, most cells possessing either IK1 (30 cells) or IK2 (24 cells) as the predominant current. 5 The characteristics of these currents suggest a possible function in the control of membrane potentials and smooth muscle quiescence in the pregnant human myometrium.

Mechanisms of the prostaglandin F2α-induced rise in [Ca2+]i in rat intrapulmonary arteries

The mechanisms by which prostaglandin F2α (PGF2α) 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 μm) of PGF2α caused a transient followed by a plateau rise in [Ca2+]i. Both responses became maximal at 0.1 μm PGF2α. At higher concentrations of PGF2α, a further slower rise in [Ca2+]i was superimposed on the plateau. The [Ca2+]i response to 0.1 μm PGF2α was mimicked by the FP receptor agonist fluprostenol, whilst the effect of 10 μm PGF2α was mimicked by the TP receptor agonist U‐46619. The plateau rise in [Ca2+]i in response to 0.1 μm PGF2α 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 μm PGF2α and 0.01 μm 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 μm PGF2α, 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 μm PGF2α was abolished, whereas 10 μm PGF2α and 0.05 μm U‐46619 still caused substantial increases. These results suggest that low concentrations of PGF2α act via FP receptors to cause IP3‐dependent Ca2+ release and store operated Ca2+ entry (SOCE). U‐46619 and 10–100 μm PGF2α 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.

Calcium antagonistic properties of the cyclooxygenase-2 inhibitor nimesulide in human myometrial myocytes

The non‐steroidal anti‐inflammatory drug nimesulide is a selective inhibitor of cyclooxygenase‐2 which relaxes spontaneously contracting human myometrium in vivo and is potentially a useful tocolytic drug. Part of the relaxant action of nimesulide may be via block of myometrial Ca2+ channels. Here, we describe the Ca2+ channel blocking properties of nimesulide in freshly dispersed human term‐pregnant myometrial smooth muscle cells (HMSMCs). Both L‐ and T‐components of the whole cell Ca2+ channel current were inhibited by 100 μM nimesulide (38±3 and 35±1% block, respectively). At physiological pH inside and outside the cell (pHo/pHi=7.4/7.2), this block did not depend on the holding or test potential, although a degree of use‐dependence was observed during high frequency stimulation at a higher concentration of drug (300 μM). At pHo/pHi=6.8, under which condition the concentration of the non‐ionized form of the drug is increased 3 fold compared to pH 7.4, nimesulide blocked the L‐type current more potently (58±3% inhibition at 100 μM, P<0.01) compared to physiological pH. Nimesulide caused a 7 mV leftward shift in the availability curve of the current at pH 6.8, suggesting that the affinity of the drug for the inactivated channel is approximately 4 fold higher than its affinity for the closed channel. We speculate that acidification and depolarization of the myometrium during the intense and prolonged contractions of labour might increase the potency of nimesulide as a Ca2+ channel antagonist, promoting its action as a tocolytic agent.

Propionate-induced relaxation in rat mesenteric arteries: a role for endothelium-derived hyperpolarising factor

Short chain fatty acids, including propionate, are generated in the caecum and large intestine, and when absorbed may elicit localised increases in intestinal blood flow. We sought to assess the mechanism by which propionate caused vasorelaxation. Propionate‐mediated relaxation of noradrenaline‐preconstricted rat mesenteric small arteries (RMSAs, i.d. 200–300 μm) was studied using small vessel myography. Propionate (1–30 mm) produced a concentration‐dependent relaxation. Relaxation induced by 10 mm propionate (the approximate EC50) was almost abolished by endothelial denudation, although a marked relaxation to a very high concentration of propionate (50 mm) persisted in the absence of the endothelium. In endothelium‐intact RMSAs, relaxation to 10 mm propionate was almost abolished by elevating [K+]o to 25 mm, but was unaffected by 100 μmNω‐nitro‐l‐arginine methyl ester (l‐NAME) (68 ± 4 vs. 66 ± 3 % in controls, n= 35), or by 1 μm indomethacin (60 ± 4 vs. 61 ± 7 % in controls, n= 15). In the presence of l‐NAME, relaxation to 10 mm propionate was significantly and markedly (i.e. > 50 %) inhibited by 50 μm Ba2+ and by the combination of 100 nm charybdotoxin and 100 nm apamin. A similar effect on propionate‐mediated relaxation was also exerted by 100 μm ouabain, and by the combination of 50 μm barium with ouabain. Relaxation was also significantly and markedly inhibited by pre‐treatment of RMSAs with 100 nm thapsigargin or 10 μm cyclopiazonic acid (CPA). The results demonstrate that 10 mm propionate relaxes RMSAs via endothelium‐derived hyperpolarising factor (EDHF). The observation that relaxation by propionate is inhibited by thapsigargin and CPA suggests that this action of propionate involves the release of endothelial cell Ca2+ stores.

Low concentrations of sphingosylphosphorylcholine enhance pulmonary artery vasoreactivity - The role of protein kinase C delta and Ca2+ entry

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC50 is ≈100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (≤1 &mgr;mol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca2+]. SPC (1 &mgr;mol/L) had no effect on force or intracellular [Ca2+] on its own, but dramatically potentiated constrictions induced by ≈25 mmol/L [K+], such that at 40 minutes, force and intracellular [Ca2+] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca2+ entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC&dgr; inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC&dgr;. The potentiation could be attributed to enhancement of Ca2+ entry. SPC also potentiated the responses to prostaglandin F2&agr; and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 &mgr;mol/L) caused translocation of PKC&dgr; to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca2+ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC&dgr;. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Divergent modulation of Rho-kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle

The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src‐family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR‐mediated ASM contraction and associated signalling events.

Tyrosine kinases as key modulators of smooth muscle function in health and disease

This issue contains two review articles based on talks given as part of a symposium entitled ‘Tyrosine kinases and smooth muscle function’ that took place in London, July 2014, hosted by the Physiological Society and supported by The Journal of Physiology. The symposium examined evidence for the involvement of non-receptor tyrosine kinases in multiple aspects of smooth muscle function, including their role in normal contractile responses to various stimuli and potentially important contributions to hyper-contractile states and vascular remodelling in hypertension. Senior speakers at the symposium were Professor Gervaise Loirand (University of Nantes, France), Professor Ralph Schermuly (Max-Plank Institute for Heart and Lung Research, Germany), and Dr Jacqueline Ohanian (Institute of Cardiovascular Sciences, University of Manchester, UK).

Role of reactive oxygen species and sulfide-quinone oxoreductase in hydrogen sulfide-induced contraction of rat pulmonary arteries

Application of H2S (“sulfide”) elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 μM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 μM) and by 50 μM ryanodine; the Ca2+ channel blocker nifedipine (1 μM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 μM), the NADPH oxidase (NOX) blocker VAS2870 (10 μM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 μM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 μM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.