S. Adriaan Nelemans

Allergic sensitization enhances the contribution of Rho-kinase to airway smooth muscle contraction

Repeated allergen challenge has been shown to increase the role of Rho‐kinase in airway smooth muscle (ASM) contraction. We considered the possibility that active allergic sensitization by itself, that is, without subsequent allergen exposure, could be sufficient to enhance Rho‐kinase‐mediated ASM contraction. Guinea pigs were actively IgE‐sensitized to ovalbumin (OA), using Al(OH)3 as adjuvant. Contractile responsiveness to Gq‐coupled receptor agonists (methacholine, histamine or PGF2α) was investigated in tracheal rings. No effect of sensitization was observed on basal‐ and methacholine‐induced myogenic tone. In contrast, potency of histamine and PGF2α increased, that is, EC50 decreased, after OA‐sensitization by 2.6‐ and 4.7‐fold, respectively, without effect on maximal contraction (Emax). Basal tone in preparations from both control and OA‐sensitized animals was strongly decreased in the presence of the Rho‐kinase inhibitor (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(4‐pyridyl) cyclohexane carboxamide (Y‐27632) (1 μM). In control preparations, the Emax and potency of histamine were unaffected by Y‐27632, but were decreased for PGF2α (by 38.2% and 2.0‐fold, respectively). However, in preparations from OA‐sensitized animals, Y‐27632 induced a significant reduction in Emax (33.5%) and potency (2.3‐fold) of histamine and of PGF2α (48.3% and 6.6‐fold, respectively), normalizing the OA‐sensitization‐induced increase in sensitivity toward these agonists. We also investigated the contribution of Rho‐kinase in vivo by measuring airway responsiveness toward inhaled histamine in permanently instrumented, unanaesthetized control and OA‐sensitized guinea pigs. Treatment with Y‐27632 by inhalation (5 mM, nebulizer concentration) decreased airway responsiveness toward histamine both in control and OA‐sensitized animals. However, the histamine PC100 ratio pre/post Y‐27632 inhalation was significantly smaller in OA‐sensitized animals as compared to control animals, indicating an enhanced contribution of Rho‐kinase. Expression of RhoA, an upstream activator of Rho‐kinase, was significantly increased (2.6‐fold) in lung homogenates of OA‐sensitized guinea pigs compared to control animals, as determined by Western analysis. In conclusion, the results show a receptor‐dependent role of Rho‐kinase in agonist‐induced ASM contraction. The contribution of Rho‐kinase to contractile airway responsiveness, both in vivo and ex vivo, is augmented after active allergic sensitization, as a consequence of increased expression of RhoA presumably. Inhibition of the RhoA/Rho‐kinase pathway may be considered a useful pharmacotherapeutical target in allergy and asthma.

Functional characterization of serum- and growth factor-induced phenotypic changes in intact bovine tracheal smooth muscle

The present study aims to investigate whether phenotypic changes, reported to occur in cultured isolated airway smooth muscle (ASM) cells, are of relevance to intact ASM. Moreover, we aimed to gain insight into the signalling pathways involved. Culturing of bovine tracheal smooth muscle (BTSM) strips for up to 8 days in the presence of 10% foetal bovine serum caused a time‐dependent (t1/2=2.8 days) decrease in maximal contraction (Emax) to methacholine compared to serum‐deprived controls (Emax=74±4% at day 8). A reduced Emax was also found using insulin‐like growth factor‐1 (30 ng ml−1) and platelet‐derived growth factor (30 ng ml−1), but not using epidermal growth factor (10 ng ml−1) (Emax=83±3, 67±8, 100±4%, respectively). Similar serum and growth factor‐induced changes in Emax were found for KCl‐induced contraction (65±9, 80±7, 64±11% and 107±2%, respectively). Strong correlations were found between the growth factor‐induced reductions in Emax and their proliferative responses, assessed by [3H]‐thymidine‐incorporation, in BTSM cells. (r=0.97, P=0.002 for methacholine and r=0.93, P=0.007 for KCl). The PDGF‐induced reduction in Emax was inhibited completely by combined treatment with either PD 98059 (30 μM) or LY 294002 (10 μM). These results indicate that serum and growth factors may cause a functional shift towards a less contractile phenotype in intact BTSM, which is associated with their proliferative response and dependent on signalling pathways involving the mitogen‐activated protein kinase pathway and the phosphatidylinositol‐3‐kinase pathway.

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

BackgroundIn addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.MethodsGrowth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM) and the selective EP1-antagonist AH-6809 (10 μM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF2α-and PGE2-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.ResultsEpidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF2α-and PGE2-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2α-and PGE2-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM) significantly reduced (approximately 50 %) and the EP1-antagonist AH-6809 (10 μM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.ConclusionThe results indicate that growth factors induce ASM contraction through contractile prostaglandins – not derived from the epithelium – which in turn rely on Rho-kinase for their contractile effects.

Δ9-Tetrahydrocannabinol activates [Ca2+]i increases partly sensitive to capacitative store refilling

Delta9-tetrahydrocannabinol induces [Ca2+]i increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+]i. Stimulation with delta9-tetrahydrocannabinol after functional downregulation of intracellular Ca2+ stores by longterm thapsigargin treatment, still induced a major Ca2+ entry and a minor Ca2+ release component. Thapsigargin sensitive influx and release were selectively inhibited by the cannabinoid CB1 receptor antagonist SR141716A. No effects on [Ca2+]i were obtained after stimulation with the CB2 receptor agonist palmitoylethanolamide. This study is the first demonstration of (1) Ca2+ release from thapsigargin sensitive intracellular stores and capacitative Ca2+ entry via CB1 receptor stimulation and of (2) an additional delta9-tetrahydrocannabinol induced thapsigargin insensitive component, mainly representing Ca2+ influx which is neither mediated by CB1 nor CB2 receptor stimulation.

Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta.

We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. Compounds were delivered into the cytoplasm of de‐endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10−5 M). Intracellular administration of Ang II (incorporation range: 0.01–300 nmol mg−1) resulted in a dose‐dependent contraction, insensitive to extracellular administration (10−6 M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non‐selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]‐Ang II (P<0.05). Intracellular administration of CV11947 or PD 123319 right shifted the dose‐response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg−1 Ang II was incorporated. Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P<0.05). Intracellular administration of Ang I and saralasin also induced contraction (P<0.05). Both responses were sensitive to intracellular CV11947 (P<0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre‐treatment with saralasin or CV11947 (P<0.05), but not with PD 123319. Extracellular saralasin induced no contraction. Intracellular Ang II induced contraction was not affected by pre‐treatment with heparin filled liposomes, but completely abolished in Ca2+‐free external medium. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+‐influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+‐stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+‐mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho‐kinase (an important regulator of Ca2+‐sensitization) to methacholine‐, pilocarpine‐ and McN‐A‐343‐induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho‐kinase inhibitor Y‐27632. In addition, we measured Ca2+‐mobilization and ‐influx in BTSM cells in response to these agonists in the absence and presence of Y‐27632. Whereas treatment with Y‐27632 (1 μM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4±2.8 and 50.4±7.9% for the partial agonists pilocarpine and McN‐A‐343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho‐kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN‐A‐343 induced a very small Ca2+‐mobilization and ‐influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho‐kinase dependency was observed. Interestingly, no inhibitory effects of Y‐27632 were observed on Ca2+‐mobilization and‐influx for all three agonists, indicating that the effects of Y‐27632 on contraction are most likely on the level of Ca2+‐sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN‐A‐343 are dependent on Rho‐kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist‐dependent role for Rho‐kinase in ASM contraction. Moreover, an inverse relationship exists between Rho‐kinase dependency and both Ca2+‐mobilization and Ca2+‐influx for these agonists.

Muscarinic M3 receptor-dependent regulation of airway smooth muscle contractile phenotype

Airway smooth muscle (ASM) cells are known to switch from a contractile to a proliferative and synthetic phenotype in culture in response to serum and growth factors. Phenotype switching in response to contractile agonists, however, is poorly characterised, despite the possible relationship between ASM phenotype and airway remodelling in asthma. To investigate the effects of muscarinic receptor stimulation on ASM phenotype, we used organ‐cultured bovine tracheal smooth muscle (BTSM) strips, in which contractile responsiveness, contractile protein expression and proliferation were measured after pretreatment with methacholine. Long‐term methacholine pretreatment (8 days) decreased maximal contraction and sensitivity to methacholine as well as to histamine and KCl. This decrease was dose‐dependent (pEC50=5.2±0.1). Pretreatment with the highest concentration of methacholine applied (100 μM) could suppress maximal histamine‐induced contraction to 8±1% of control. In addition, contractile protein expression (myosin, actin) was downregulated two‐fold. No concomitant increase in proliferative capacity was observed. The M3/M2 muscarinic receptor antagonist DAU 5884 (0.1 μM) completely inhibited the observed decrease in contractility. In contrast, the M2/M3 muscarinic receptor antagonist gallamine (10 μM) was ineffective, demonstrating that M2 receptors were not involved. Pretreatment (8 days) with 60 mM KCl could mimick the strong decreases in contractility. This was completely prevented by pretreatment with verapamil (1 μM). Regulation of contractility was not affected by protein kinase C inhibition, whereas inhibitors of phosphatidyl inositol 3‐kinase and p42/p44 mitogen activated protein kinase were partially effective. These results show that long‐term methacholine pretreatment (8 days) induces an M3 receptor‐dependent decrease in BTSM contractility without increased proliferative capacity.

Microarray profiling of lymphocytes in internal diseases with an altered immune response, potential and methodology

Recently it has become possible to investigate expression of all human genes with microarray technique. The authors provide arguments to consider peripheral white blood cells and in particular lymphocytes as a model for the investigation of pathophysiology of asthma, RA, and SLE diseases in which inflammation is a major component. Lymphocytes are an alternative to tissue biopsies that are most often difficult to collect systematically. Lymphocytes express more than 75% of the human genome, and, being an important part of the immune system, they play a central role in the pathogenesis of asthma, RA, and SLE. Here we review alterations of gene expression in lymphocytes and methodological aspects of the microarray technique in these diseases. Lymphocytic genes may become activated because of a general nonspecific versus disease-specific mechanism. The authors suppose that in these diseases microarray profiles of gene expression in lymphocytes can be disease specific, rather than inflammation specific. Some potentials and pitfalls of the array technologies are discussed. Optimal clinical designs aimed to identify disease-specific genes are proposed. Lymphocytes can be explored for research, diagnostic, and possible treatment purposes in these diseases, but their precise value should be clarified in future investigation.

Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells.

Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT(1) receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPbetaS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.

Normalization of aortic function during arousal episodes in the hibernating ground squirrel

Hypothermia is commonly used to restrict organ damage during preservation of tissue, but does not offer complete protection. Organ damage after reperfusion/rewarming is amongst others caused by an impairment of vascular properties, particularly endothelium-dependent vasodilatation. We hypothesized that hibernating small animals, which frequently cycle through periods of deep cooling (torpor) and full rewarming (arousal), employ specific mechanisms to preserve vascular function after cooling and reperfusion. Therefore we measured contraction of aortic tissue of hibernating European ground squirrels after 24 h and 7 days of torpor, arousal (1.5 h) and in non-hibernating animals. To assess the role of nitric oxide (NO), experiments were performed in the absence and presence of the NO-synthesis inhibitor, L-NMMA (10(-4) M). Maximum contraction to phenylephrine and angiotensin II was doubled in 7-days torpid animals without a shift in EC50, compared to the other 3 groups. Maximum contraction to KCl was doubled in 7-days torpid animals compared to the arousal group and non-hibernating animals. Relaxation to acetylcholine (ACh) and sodium nitrite in phenylephrine precontracted rings did not differ between groups. In the presence of L-NMMA, the maximum of concentration-response curves for all three vasoconstrictors was increased by about 30% in the arousal group, but unaffected in other groups. L-NMMA completely inhibited ACh-induced relaxation in 24-h torpid animals and non-hibernating animals, but only partially in 7-days torpid animals and in the arousal group. From this we conclude that vascular adaptation proceeds during torpor. Further, increased contractility of aortic tissue during long torpor returns to normal within 1.5 hours of arousal, which is associated with an increased basal NO synthesis. In addition, involvement of NO in agonist-mediated relaxation differs between the various stages of hibernation.Thus, hibernating animals have effectively developed mechanisms to preserve vascular function after cooling and rewarming.