Christopher N. Fortner

Aquaporin 5-Deficient Mouse Lungs are Hyperresponsive to Cholinergic Stimulation

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5−/−) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5+/+ mice, Aqp5−/− mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5−/− mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5−/− mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.

ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

Zipper-interacting protein kinase (ZIPK) regulates Ca2+-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.

Transgenic overexpression of beta(2)-adrenergic receptors in airway smooth muscle alters myocyte function and ablates bronchial hyperreactivity.

β2-Adrenergic receptors (β2AR) act to relax airway smooth muscle and can serve to counteract hyperresponsiveness, although the effect may not be ablative even in the presence of exogenous agonist. Within this signaling cascade that ultimately transduces smooth muscle relaxation, a significant “spare receptor” pool has been hypothesized to be present in the airway. In order to modify the relationship between β2AR and downstream effectors, transgenic mice (TG) were created overexpressing β2AR ∼75-fold in airway smooth muscle using a mouse smooth muscle α-actin promoter. While >90% of these receptors were expressed on the smooth muscle cell surface, the percentage of receptors able to form the agonist-promoted high affinity complex was less than that found with nontransgenic (NTG) cells (R H = 18 versus 36%). Nevertheless, β2AR signaling was found to be enhanced. Intact airway smooth muscle cells from TG had basal cAMP levels that were greater than NTG cells. A marked increase in agonist-stimulated cAMP levels was found in the TG (∼200% stimulation over basal) compared with NTG (∼50% over basal) cells. Adenylyl cyclase studies gave similar results and also showed a 10-fold lower EC50for TG cells. Tracheal rings from TG mice that were precontracted with acetylcholine had an enhanced responsiveness (relaxation) to β-agonist, with a 60-fold decrease in the ED50, indicating that the enhanced signaling imposed by overexpression results in an increase in the coordinated function of the intact airway cells. In vivo studies showed a significantly blunted airway resistance response to the inhaled bronchoconstrictor methacholine in the TG mice. Indeed, with β-agonist pretreatment, the TG mice displayed no response whatsoever to methacholine. These results are consistent with β2AR being the limiting factor in the transduction system. Increases in the initial component of this transduction system (the β2AR) are sufficient to markedly alter signaling and airway smooth muscle function to the extent that bronchial hyperresponsiveness is ablated, consistent with an anti-asthma phenotype.

Angiotensin II Type 1A Receptors in Vascular Smooth Muscle Cells Do Not Influence Aortic Remodeling in Hypertension

Vascular injury and remodeling are common pathological sequelae of hypertension. Previous studies have suggested that the renin-angiotensin system acting through the type 1 angiotensin II (AT1) receptor promotes vascular pathology in hypertension. To study the role of AT1 receptors in this process, we generated mice with cell-specific deletion of AT1 receptors in vascular smooth muscle cells using Cre/Loxp technology. We crossed the SM22&agr;-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Agtr1a gene (Agtr1aflox), encoding the major murine AT1 receptor isoform (AT1A). In SM22&agr;-Cre+Agtr1aflox/flox (SMKO) mice, AT1A receptors were efficiently deleted from vascular smooth muscle cells in larger vessels but not from resistance vessels such as preglomerular arterioles. Thus, vasoconstrictor responses to angiotensin II were preserved in SMKO mice. To induce hypertensive vascular remodeling, mice were continuously infused with angiotensin II for 4 weeks. During infusion of angiotensin II, blood pressures increased significantly and to a similar extent in SMKO and control mice. In control mice, there was evidence of vascular oxidative stress indicated by enhanced nitrated tyrosine residues in segments of aorta; this was significantly attenuated in SMKO mice. Despite these differences in oxidative stress, the extent of aortic medial expansion induced by angiotensin II infusion was virtually identical in both groups. Thus, vascular AT1A receptors promote oxidative stress in the aortic wall but are not required for remodeling in angiotensin II–dependent hypertension.

Ablation of the SERCA3 gene alters epithelium-dependent relaxation in mouse tracheal smooth muscle

Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3), an isoform of the intracellular Ca2+ pump that has been shown to mediate endothelium-dependent relaxation of vascular smooth muscle, is also expressed in tracheal epithelium. To determine its possible role in regulation of airway mechanical function, we compared tracheal contractility in gene-targeted mice deficient in SERCA3 (SERCA3-) with that in wild-type tracheae. Cumulative addition of ACh elicited concentration-dependent increases in isometric force (ED50 = 2 μM, maximum force = 8 mN/mm2) that were identical in SERCA3- and wild-type tracheae. After ACh stimulation, substance P (SP) elicited a transient relaxation (42.6 ± 3.2%, n = 28) in both tracheae. However, the rate of relaxation was significantly ( P < 0.04, n = 9) more rapid in the wild-type [half-time ( t ½) = 34.3 s] than in the SERCA3-( t ½ = 61.6 s) trachea. The SP relaxation was reduced by rubbing the trachea, indicative of epithelial cell involvement. This was verified using a perfused trachea preparation. SP in the outside medium had no effect, whereas SP in the perfusate bathing the epithelial side elicited a relaxation. Nitric oxide synthase inhibition (0.2 mM N ω-nitro-l-arginine) reduced the SP relaxation by 36.5 ± 12.5%, whereas the SP effect was abolished by eicosanoid inhibition (10 μM indomethacin). ATP also elicited an epithelium-dependent relaxation similar to SP but with a more rapid relaxation in the SERCA3-trachea than in the wild-type trachea. Our results indicate that SERCA3 gene ablation does not directly affect smooth muscle, which is consistent with the distribution of the isoform, but suggest that SERCA3 plays a role in epithelial cell modulation of airway smooth muscle function.

Deletion of the Protein Kinase A/Protein Kinase G Target SMTNL1 Promotes an Exercise-adapted Phenotype in Vascular Smooth Muscle

In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1-/- mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to α-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1-/- mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1+/+ mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.

Fluorescence Linked Enzyme Chemoproteomic Strategy for Discovery of a Potent and Selective DAPK1 and ZIPK Inhibitor

DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.

Smoothelin-like 1 Protein Regulates Myosin Phosphatase-targeting Subunit 1 Expression during Sexual Development and Pregnancy

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30–40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.