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 ␣-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.The contractile state of smooth muscle (SM) 2 is largely governed by phosphorylation of myosin regulatory light chain (LC20), which in turn is regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase, SMPP1M (1). In SM, both MLCK and SMPP1M are regulated, thereby enabling homeostatic control of contractile activity (2). Exquisite control is necessary because of the essential roles of SM in many physiological processes, such as maintenance of blood pressure. In most SMs, release of cyclic nucleotides (cGMP/cAMP), in response to hormonal or neuronal stimulation, promote relaxation either by lowering intracellular [Ca 2ϩ ] or desensitizing the muscle to Ca 2ϩ by inhibiting MLCK and/or activating SMPP1M (2).Targeted deletions of both PKA and PKG in mice produce profound phenotypes, underscoring the importance of these kinases in many physiological processes (3, 4). In vivo, both kinases are known to selectively target a discrete number of substrates and current thinking suggests that selective targeting is the means by which these broadly acting enzymes bring about coordinated physiological responses (5). A few groups have begun to test this hypothesis by selectively deleting PKA/PKG targets in mice. Schlossmann et al. (6) demonstrated a major role for IRAG (inositol 1,4,5-trisphosphate receptor 1 IP3R1-associated protein, with exon 12 deleted by removing the 1,4,5-trisphosphate receptor binding domain) in PKG-mediated regulation of [Ca 2ϩ ] in SM. Disruption of IRAG resulted in a selective loss of signaling response. Other PKG/PKA-mediated responses were largely intact, contrasting wi...

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 and rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca2+-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.

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.

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.A quaporin 5 (AQP5) is a mercury-sensitive water channel expressed in mouse salivary gland acinar cells, alveolar type I cells, lacrimal glands, and the eye (1-3). However, the function of AQP5 in mouse lung physiology and pathophysiology is largely unknown. Recent studies have explored the regulation of Aqp5 gene expression in normal and disease states. In a mouse model of acute lung injury, AQP5 mRNA and protein expression were significantly decreased in association with pulmonary inflammation and edema resulting from adenoviral infection (4). Moreover, AQP5 expression in a murine lung epithelial cell line (MLE-12) is modulated by the proinf lammatory cytokine TNF-␣ through an nuclear factor (NF)-B-dependent pathway (5). Keratinocyte growth factor also has been shown to mediate the differentiation status of alveolar epithelial cells and AQP5 expression (6). However, a clear understanding of AQP5 function in the lung remains to be discerned.Previously published studies have addressed functional questions limited to the examination of the role of AQP5 in type I cells in fluid clearance and airspace-capillary osmotic water permeability. Of significance, isolated perfused lungs from Aqp5 Ϫ/Ϫ mice were shown to have a 10-fold reduction in airspace-capillary osmotic water permeability (7). Experiments addressing the role of AQP5 in alveolar fluid clearance failed to indicate a role for AQP5 in this process under certain physiological conditions (8). Based on the results of these studies, some have concluded that AQP5 does not play a major role in lung physiology. Contrary to these conclusions, the data reported here clearly define a...

Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2 from airway epithelium in other species, suggesting PGE2 as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2 receptor [EP2(-/-)], one of the PGE2 receptors, we tested the hypothesis that PGE2 is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(-/-) mice. There were no differences between EP2(-/-) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(-/-) and wild-type tracheas in basal or stimulated PGE2 production. Exogenous PGE2 produced significantly less relaxation in EP2(-/-) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2 receptors are of primary importance in airway relaxation to PGE2 and relaxation to SP or ATP is mediated through PGE2 acting on EP2 receptors.

␤ 2 -Adrenergic receptors (␤ 2 AR) 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 ␤ 2 AR and downstream effectors, transgenic mice (TG) were created overexpressing ␤ 2 AR ϳ75-fold in airway smooth muscle using a mouse smooth muscle ␣-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, ␤ 2 AR 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 (ϳ200% stimulation over basal) compared with NTG (ϳ50% over basal) cells. Adenylyl cyclase studies gave similar results and also showed a 10-fold lower EC 50 for TG cells. Tracheal rings from TG mice that were precontracted with acetylcholine had an enhanced responsiveness (relaxation) to ␤-agonist, with a 60-fold decrease in the ED 50 , 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 ␤-agonist pretreatment, the TG mice displayed no response whatsoever to methacholine. These results are consistent with ␤ 2 AR being the limiting factor in the transduction system. Increases in the initial component of this transduction system (the ␤ 2 AR) are sufficient to markedly alter signaling and airway smooth muscle function to the extent that bronchial hyperresponsiveness is ablated, consistent with an antiasthma phenotype.

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.

Abstract-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 (AT 1 ) receptor promotes vascular pathology in hypertension. To study the role of AT 1 receptors in this process, we generated mice with cell-specific deletion of AT 1 receptors in vascular smooth muscle cells using Key Words: angiotensin II Ⅲ hypertrophy Ⅲ hyperplasia Ⅲ aorta Ⅲ smooth muscle Ⅲ hypertension T he renin-angiotensin system (RAS) is a principal regulator of blood pressure homeostasis; dysregulation of this system commonly contributes to human hypertension. 1,2 Accordingly, pharmacological inhibitors of the RAS, including angiotensin converting enzyme inhibitors and angiotensin receptor blockers, can effectively lower blood pressure in a significant proportion of patients with essential hypertension. 3,4 Moreover, these agents also attenuate end-organ damage, decreasing cardiovascular morbidity, and slowing the progression of chronic kidney injury. 5,6 It has been suggested that RAS inhibitors provide protection against complications of hypertension beyond their effects to lower blood pressure, indicating nonhemodynamic, cellular actions of angiotensin II to promote tissue damage. 7 However, in some clinical trials, end-organ protection by RAS inhibition has been accompanied by more effective reduction of blood pressure. 4,8,9 Moreover, studies in animal models have suggested that the antihypertensive actions of RAS inhibitors are critical for preventing cardiac hypertrophy 10 and progressive kidney injury. 11 The vascular system is a major target of damage in hypertension. Expansion of arteries and arterioles in the kidney, also called nephrosclerosis, is the most common renal pathological lesion accompanying hypertension 12 and is an important cause of chronic kidney disease in blacks. 13 Vessel remodeling with changes in compliance is also seen in the aorta and other vascular beds in hypertension, 14 in which the RAS has potent actions to influence vascular structure and function. 15 For example, angiotensin II causes systemic vasoconstriction by activa-