Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.

NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations.

Inflammasomes are positioned to rapidly escalate the intensity of inflammation by activating interleukin (IL)-1ß, IL-18 and cell death by pyroptosis. However, negative regulation of inflammasomes remains poorly understood, as is the signaling cascade that dampens inflammasome activity. We found that rapid NLRP3 inflammasome activation was directly inhibited by protein kinase A (PKA), which was induced by prostaglandin E2 (PGE2) signaling via the PGE2 receptor E-prostanoid 4 (EP4). PKA directly phosphorylated the cytoplasmic receptor NLRP3 and attenuated its ATPase function. We found that Ser295 in human NLRP3 was critical for rapid inhibition and PKA phosphorylation. Mutations in NLRP3-encoding residues adjacent to Ser295 have been linked to the inflammatory disease CAPS (cryopyrin-associated periodic syndromes). NLRP3-S295A phenocopied the human CAPS mutants. These data suggest that negative regulation at Ser295 is critical for restraining the NLRP3 inflammasome and identify a molecular basis for CAPS-associated NLRP3 mutations.

Extracellular cathepsin Z signals through the α5 integrin and augments NLRP3 inflammasome activation.

Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1ß production. Cathepsin Z has been associated with the development of inflammation and IL-1ß production; however, the mechanism of how cathepsin Z leads to IL-1ß production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1ß release was determined using enzyme-linked immunosorbent assay or IL-1ß bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1ß expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1ß through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.

Death-associated protein kinase 3 regulates the myogenic reactivity of cerebral arteries.

NEW FINDINGS: What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. ABSTRACT: The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

Smoothelin-like 1 knockout mice display sex-dependent alterations in blood flow and cardiac function.

Smoothelin-like 1 (SMTNL1) modulates the contractile performance of smooth muscle and thus has a key role in vascular homeostasis. Elevated vascular tone, recognized as a contributor to the development of progressive cardiac dysfunction, was previously found with SMTNL1 deletion. In this study, we assessed cardiac morphology and function of male and female, wild-type (Smtnl1+/+) and global SMTNL1 knockout (Smtnl1-/-) mice at 10 weeks of age. Gross dissection revealed distinct cardiac morphology only in males; Smtnl1-/- hearts were significantly smaller than Smtnl1+/+, but the left ventricle (LV) proportion of heart mass was greater. Male Smtnl1-/- mice also displayed increased ejection fraction and fractional shortening, as well as elevated aortic and pulmonary flow velocities. The impact of cardiac stress with pressure overload by transverse aortic constriction (TAC) was examined in male mice. With TAC banding, systolic function was preserved, but the LV filling pressure was selectively elevated due to relaxation impairment. Smtnl1-/- mice displayed higher early/passive filling velocity of LV/early mitral annulus velocity ratio (E/E' ratio) and myocardial performance index along with a prolonged isovolumetric relaxation time. Taken together, the findings support a novel, sex-dimorphic role for SMTNL1 in modulating cardiac structure and function of mice.

WOMAC score and arthritis diagnosis predict decreased agricultural productivity.

BACKGROUND: Arthritis and joint pain are highly prevalent in agricultural (ag) workers. Many ag operations are sustained by a small number of workers, and the disability of even one worker thus contributes to economic hardship. This study investigated associations between joint health in Montana ag workers and economic well-being and work capacity. METHODS: This observational mixed-methods study utilized quantitative survey data and qualitative focus group data. 299 ranchers and farmers in 9 Montana counties completed either an online or paper survey that included participant demographics, joint symptoms, history of arthritis and arthritis type, financial status, work capacity, and the need to rely on others to complete one's work. The Western Ontario and McMaster Universities arthritis index (WOMAC) survey was completed by those with hip or knee pain. Data were entered into REDCap v8.9.2 for analysis with SAS 9.4, using logistic and linear regression models to detect associations between covariables and to calculate odds ratios and confidence intervals. Focus groups were held with ranchers in two Montana counties, discussing similar topics, and the themes expressed were identified. RESULTS: 87.6% of survey respondents reported joint pain, 47.8% a diagnosis of arthritis, and 22.4% osteoarthritis (OA). A 10-point increase in WOMAC was significantly associated with lower work capacity (OR 2.00; 95% CI [1.58, 2.55], p < 0.01), worse financial condition (OR 1.23; 95% CI [1.01,1.48], p = 0.04), and increased reliance on others (OR 1.82; 95% CI [1.32, 2.55], p < 0.01). An arthritis diagnosis was associated with worsening work capacity (OR 4.66; 95% CI [2.71, 8.01], p < 0.01) and increased odds of relying on others (OR 3.23; 95% CI [1.56, 6.66], p < 0.01). A diagnosis of OA was significantly associated with decreased work capacity (OR 3.47; 95% CI [1.97, 6.11], p < 0.01). Unadjusted for age and BMI, we found a significant association between years spent working in agriculture and joint health, which became non-significant after adjusting for age and BMI. Focus group themes included decreased productivity with increased joint symptoms and a tendency for ranchers to avoid interaction with the health care system. CONCLUSION: Poor joint health is associated with economic risk on Montana ranches and farms.

Toward a Better Understanding of In-Vehicle Auditory Warnings and Background Noise.

OBJECTIVE: The effectiveness of three types of in-vehicle warnings was assessed in a driving simulator across different noise conditions. BACKGROUND: Although there has been much research comparing different types of warnings in auditory displays and interfaces, many of these investigations have been conducted in quiet laboratory environments with little to no consideration of background noise. Furthermore, the suitability of some auditory warning types, such as spearcons, as car warnings has not been investigated. METHOD: Two experiments were conducted to assess the effectiveness of three auditory warnings (spearcons, text-to-speech, auditory icons) with different types of background noise while participants performed a simulated driving task. RESULTS: Our results showed that both the nature of the background noise and the type of auditory warning influenced warning recognition accuracy and reaction time. Spearcons outperformed text-to-speech warnings in relatively quiet environments, such as in the baseline noise condition where no music or talk-radio was played. However, spearcons were not better than text-to-speech warnings with other background noises. Similarly, the effectiveness of auditory icons as warnings fluctuated across background noise, but, overall, auditory icons were the least efficient of the three warning types. CONCLUSION: Our results supported that background noise can have an idiosyncratic effect on a warning's effectiveness and illuminated the need for future research into ameliorating the effects of background noise. APPLICATION: This research can be applied to better present warnings based on the anticipated auditory environment in which they will be communicated.

Chemical Modulation of the 1-(Piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one Scaffold as a Novel NLRP3 Inhibitor.

In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1ß release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1ß release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein-ligand binding that might explain the activity of the compounds.

ATP-Binding and Hydrolysis in Inflammasome Activation.

The prototypical model for NOD-like receptor (NLR) inflammasome assembly includes nucleotide-dependent activation of the NLR downstream of pathogen- or danger-associated molecular pattern (PAMP or DAMP) recognition, followed by nucleation of hetero-oligomeric platforms that lie upstream of inflammatory responses associated with innate immunity. As members of the STAND ATPases, the NLRs are generally thought to share a similar model of ATP-dependent activation and effect. However, recent observations have challenged this paradigm to reveal novel and complex biochemical processes to discern NLRs from other STAND proteins. In this review, we highlight past findings that identify the regulatory importance of conserved ATP-binding and hydrolysis motifs within the nucleotide-binding NACHT domain of NLRs and explore recent breakthroughs that generate connections between NLR protein structure and function. Indeed, newly deposited NLR structures for NLRC4 and NLRP3 have provided unique perspectives on the ATP-dependency of inflammasome activation. Novel molecular dynamic simulations of NLRP3 examined the active site of ADP- and ATP-bound models. The findings support distinctions in nucleotide-binding domain topology with occupancy of ATP or ADP that are in turn disseminated on to the global protein structure. Ultimately, studies continue to reveal how the ATP-binding and hydrolysis properties of NACHT domains in different NLRs integrate with signaling modules and binding partners to control innate immune responses at the molecular level.

An algorithm to minimize the number of blocks in incomplete block designs.

Incomplete block designs are experimental designs in which a subset of treatments are included in each block. The researcher must decide which conditions are assigned to each block. This design concept is quite general. At the level of the experiment, a treatment is a condition in an experiment, blocks are different groups of subjects, and the researcher must decide how to assign a subset of conditions to each block of subjects. At the level of the subject, the treatments correspond to individual stimuli, blocks correspond to experimental trials, and the researcher must decide which subset of stimuli to include in each trial. In this article, we present an efficient algorithm that assigns treatments to blocks in an incomplete block design according to two criteria: Each pair of treatments must appear together in at least one block, and the number of blocks in the experiment is minimized. We discuss details and applications of the algorithm and provide software and a web application to generate designs according to the needs of the researcher.

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening.

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Pregnane X Receptor Activation Triggers Rapid ATP Release in Primed Macrophages That Mediates NLRP3 Inflammasome Activation.

The pregnane X receptor (PXR) is a ligand-activated nuclear receptor that acts as a xenobiotic sensor, responding to compounds of foreign origin, including pharmaceutical compounds, environmental contaminants, and natural products, to induce transcriptional events that regulate drug detoxification and efflux pathways. As such, the PXR is thought to play a key role in protecting the host from xenobiotic exposure. More recently, the PXR has been reported to regulate the expression of innate immune receptors in the intestine and modulate inflammasome activation in the vasculature. In the current study, we report that activation of the PXR in primed macrophages triggers caspase-1 activation and interleukin-1ß release. Mechanistically, we show that this response is nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3-dependent and is driven by the rapid efflux of ATP and P2X purinoceptor 7 activation following PXR stimulation, an event that involves pannexin-1 gating, and is sensitive to inhibition of Src-family kinases. Our findings identify a mechanism whereby the PXR drives innate immune signaling, providing a potential link between xenobiotic exposure and the induction of innate inflammatory responses.

Effects of phosphorylation on the NLRP3 inflammasome.

The pyrin domain containing Nod-like receptors (NLRPs) are a family of pattern recognition receptors known to regulate an array of immune signaling pathways. Emergent studies demonstrate the potential for regulatory control of inflammasome assembly by phosphorylation, notably NLRP3. Over a dozen phosphorylation sites have been identified for NLRP3 with many more suggested by phosphoproteomic studies of the NLRP family. Well characterized NLRP3 phosphorylation events include Ser198 by c-Jun terminal kinase (JNK), Ser295 by protein kinase D (PKD) and/or protein kinase A (PKA), and Tyr861 by an unknown kinase but is dephosphorylated by protein tyrosine phosphatase non-receptor 22 (PTPN22). Since the PKA- and PKD-dependent phosphorylation of NLRP3 at Ser295 is best characterized, we provide detailed review of this aspect of NLRP3 regulation. Phosphorylation of Ser295 can attenuate ATPase activity as compared to its dephosphorylated counterpart, and this event is likely unique to NLRP3. In silico modeling of NLRP3 is useful in predicting how Ser295 phosphorylation might impact upon the structural topology of the ATP-binding domain to influence catalytic activity. It is important to gain as complete understanding as possible of the complex phosphorylation-mediated mechanisms of regulation for NLRP3 in part because of its involvement in many pathological processes.

Application of immobilized ATP to the study of NLRP inflammasomes.

The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.

Validation of chemical genetics for the study of zipper-interacting protein kinase signaling.

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr kinase that mediates a variety of cellular functions. Analogue-sensitive kinase technology was applied to the study of ZIPK signaling in coronary artery smooth muscle cells. ZIPK was engineered in the ATP-binding pocket by substitution of a bulky gatekeeper amino acid (Leu93) with glycine. Cell-permeable derivatives of pyrazolo[3,4-d]pyrimidine provided effective inhibition of L93G-ZIPK (1NM-PP1, IC50 , 1.0 µM; 3MB-PP1, IC50 , 2.0 µM; and 1NA-PP1, IC50 , 8.6 µM) but only 3MB-PP1 had inhibitory potential (IC50 > 10 µM) toward wild-type ZIPK. Each of the compounds also attenuated Rho-associated coiled-coil containing protein kinase (ROCK) activity under experimental conditions found to be optimal for inhibition of L93G-ZIPK. In silico molecular simulations showed effective docking of 1NM-PP1 into ZIPK following mutational enlargement of the ATP-binding pocket. Molecular simulation of 1NM-PP1 docking in the ATP-binding pocket of ROCK was also completed. The 1NM-PP1 inhibitor was selected as the optimal compound for selective chemical genetics in smooth muscle cells since it displayed the highest potency for L93G-ZIPK relative to WT-ZIPK and the weakest off-target effects against other relevant kinases. Finally, the 1NM-PP1 and L93G-ZIPK pairing was effectively applied in vascular smooth muscle cells to manipulate the phosphorylation level of LC20, a previously defined target of ZIPK.

Quantitation of myosin regulatory light chain phosphorylation in biological samples with multiple reaction monitoring mass spectrometry.

The 20-kDa regulatory light chain of myosin II plays an important role in regulating smooth muscle contractile force. LC20 is phosphorylated canonically by myosin light chain kinase in a Ca2+/calmodulin-dependent manner at S19. The diphosphorylation of LC20 at T18 and S19 has been observed in smooth muscle tissues. Given that the phosphorylation of LC20 is positively correlated with tension development, the molar stoichiometry of LC20 phosphorylation is commonly profiled as a measure of smooth muscle contractility. Herein, we describe a novel multiple reaction monitoring (MRM)-mass spectrometry (MS) approach for the quantification of LC20 phosphorylation at T18 and S19. Unique precursor as well as y- and b-ion transitions were identified for unphosphorylated LC20-(TS), monophosphorylated LC20-(TpS) and diphosphorylated LC20-(pTpS) peptides. The MRM-MS assay could accurately define molar phosphorylation stoichiometries of S19 and T18 over a broad range (i.e., 0-2 mol P/mol LC20). Correlations of the results for two quantification techniques indicate that the MRM-MS assay performs equally to Phos-tag SDS-PAGE for the determination of LC20 phosphorylation stoichiometry in arterial tissue samples. The MRM-MS technique provides a robust alternative to antibody-based detection systems for the quantification of LC20 phosphorylation.

Effectiveness of mindfulness-based stress reduction in a community sample over 2 years.

BACKGROUND: Although numerous studies have demonstrated that mindfulness-based stress reduction (MBSR) improves clinical and nonclinical outcomes, few studies have followed MBSR participants for >12 months, and few have analyzed post-intervention home practice. METHODS: We followed a community sample of 247 self-referred adults for 2 years after completion of an 8-week MBSR program. Dependent variables, including self-reported anxiety, depression, perceived wellness, mindfulness, and duration and frequency of continued home practice, were measured before and after the program and every 6 months after. RESULTS: Participants reported significantly improved symptoms of depression and anxiety, increased perceived wellness, and increased mindfulness after the 8-week intervention compared with preintervention reports. These improvements persisted for 2 years. Correlations between post-intervention home practice and mindfulness indicated that individuals who continued to practice developed greater mindfulness, which was associated with improved mental health and wellness. CONCLUSIONS: Participants who completed the MBSR program reported significant improvements in anxiety, depression, perceived wellness, and mindfulness during the program.

Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation.

BACKGROUND: The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated. RESULTS: Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in K D value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium. CONCLUSIONS: Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.

Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

Phosphorylation of the myosin-targeting subunit 1 of myosin light chain phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr(697)and Thr(855), PKA phosphorylates these two sites and the neighboring Ser(696)and Ser(854), and prior phosphorylation at Thr(697)and Thr(855)by ROCK precludes phosphorylation at Ser(696)and Ser(854)by PKA. Furthermore, phosphorylation at Thr(697)and Thr(855)by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.