Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca 2؉ sensitization/desensitization in smooth muscle. Ca 2؉ sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca 2؉ desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca 2؉ sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca 2؉ desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.Contraction and relaxation of smooth muscle are primarily determined by the level of phosphorylation of the myosin light chain .

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...

A controlled, blind study was conducted to define the initial inflammatory response and lung damage associated with the death of precardiac stages of Dirofilaria immitis in cats as compared to adult heartworm infections and normal cats. Three groups of six cats each were used: UU: uninfected untreated controls; PreS I: infected with 100 D. immitis L3 by subcutaneous injection and treated topically with selamectin 32 and 2 days pre-infection and once monthly for 8 months); IU: infected with 100 D. immitis L3 and left untreated. Peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected from all cats on Days 0, 70, 110, 168, and 240. CT images were acquired on Days 0, 110, and 240. Cats were euthanized, and necropsies were conducted on Day 240 to determine the presence of heartworms. Bronchial rings were collected for in vitro reactivity. Lung, heart, brain, kidney, and liver tissues were collected for histopathology. Results were compared for changes within each group. Pearson and Spearman correlations were performed for association between histologic, radiographic, serologic, hematologic and bronchoalveolar lavage (BAL) results. Infected cats treated with selamectin did not develop radiographically evident changes throughout the study, were heartworm antibody negative, and were free of adult heartworms and worm fragments at necropsy. Histologic lung scores and CT analysis were not significantly different between PreS I cats and UU controls. Subtle alveolar myofibrosis was noted in isolated areas of several PreS I cats and an eosinophilic BAL cytology was noted on Days 75 and 120. Bronchial ring reactivity was blunted in IU cats but was normal in PreS I and UU cats. The IU cats became antibody positive, and five cats developed adult heartworms. All cats with heartworms were antigen positive at one time point; but one cat was antibody positive, antigen negative, with viable adult females at necropsy. The CT revealed early involvement of all pulmonary arteries and a random pattern of parenchymal disease with severe lesions immediately adjacent to normal areas. Analysis of CT 3D reconstruction and Hounsfield units demonstrated lung disease consistent with restrictive pulmonary fibrosis with an interstitial infiltrate, absence of air trapping, and decrease in total lung volume in Group IU as compared to Groups UU and PreS I. The clinical implications of this study are that cats pretreated with selamectin 1 month before D. immitis L3 infection did not become serologically positive and did not develop pulmonary arterial hypertrophy and myofibrosis.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and β subunits separately (TSD α + β) injected at high (1.3 × 10 vector genomes) or low (4.2 × 10 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + β sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + β), and ganglioside clearance was most widespread in the TSD α + β high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

Background: Results of diagnostic tests for equine pituitary pars intermedia dysfunction (PPID), including endogenous ACTH concentration and the overnight dexamethasone suppression test (DST), are affected by season. New and potentially more sensitive diagnostic tests for equine PPID, such as thyrotropin-releasing hormone (TRH)-stimulated ACTH response, have been developed, but have had limited evaluation of seasonality.Objective: Our purpose was to evaluate seasonal changes in plasma ACTH and alpha-melanocyte-stimulating hormone (a-MSH) responses to TRH administration.Animals: Nine, healthy, aged horses with normal DST results. Methods: Synthetic TRH (1 mg) was administered IV. Plasma ACTH and a-MSH concentrations were measured at 0,5, 10,15,20, 25,30, 45, 60, and 180 minutes. Testing was performed in February, July, August, September, October, and November. Mean TRH-stimulated ACTH and a-MSH concentrations were compared across months and time by repeated measures analysis of variance. Significance was set at the P o .05 level.Results: Concentrations of ACTH and a-MSH significantly increased after TRH administration. Endogenous and TRHstimulated ACTH and a-MSH concentrations were significantly different across months with higher concentrations in the summer and fall compared with February.Conclusions and Clinical Importance: Plasma ACTH and a-MSH responses to TRH administration experience seasonal variation, with TRH-stimulated ACTH and a-MSH concentrations increasing from summer through fall. These results support previous evidence of a seasonal influence on the equine pituitary-adrenal axis. More research is warranted with a larger number of horses to determine if seasonal reference ranges for TRH stimulation testing need to be defined.

BackgroundIncreased free cortisol fraction is associated with insulin dysregulation (ID) in people with Metabolic Syndrome and Cushing's Disease. Free cortisol has not been investigated in equine endocrine disorders.Hypotheses(1) In healthy horses, sex, age, body condition score (BCS), and season impact free cortisol; (2) free cortisol is increased in horses with Pituitary Pars Intermedia Dysfunction (PPID) or Equine Metabolic Syndrome (EMS).AnimalsFifty‐seven healthy horses; 40 horses and ponies with PPID (n = 20) or EMS (n = 20).MethodsProspective study. Serum collected seasonally from healthy animals and archived serum from PPID and EMS animals was analyzed for insulin, total and free cortisol concentrations, and free cortisol fraction (FCF). Linear mixed models were used to determine effects of age, sex, season, and BCS on hormones in controls. Hormone measurements were compared between disease groups and age‐ and season‐matched controls with t‐tests. EMS and hyperinsulinemic PPID animals were combined in an ID (hyperinsulinemia) group.ResultsFree cortisol concentrations were increased in overweight/obese controls (0.3 ± 0.1 μg/dL) compared to lean controls (0.2 ± 0.1 μg/dL; P = .017). Mean FCF was significantly higher in animals with PPID (8.8 ± 5.8 μg/dL, P = .005) or ID (8.8 ± 10.2 μg/dL, P = .039) than controls (5.0 ± 0.9 μg/dL), but total cortisol concentrations were similar (P ≥ .350) (PPID: 4.2 ± 4.3 μg/dL; ID: 5.0 ± 4.5 μg/dL; controls: 4.6 ± 1.7 and 5.1 ± 2.1 μg/dL).Conclusions and Clinical ImportanceIncreased FCF is associated with obesity in healthy horses and with ID (hyperinsulinemia) in horses and ponies with endocrine disease. Decreased plasma cortisol‐binding capacity could be a component of these endocrine disorders in horses.

This study presents clinical findings after oral ingestion of Toxocara cati eggs which resulted in rapid pulmonary lung migration and parenchymal disease, noted on clinically relevant diagnostic methods. Further, the study investigated the efficacy of pre-infection applications of preventative medication on larval migration through the lungs. A third aim of the study was to determine if adult cats infected with T. cati developed lung disease. Cats in infected groups were administered five oral doses of L3 T. cati larvae. Four-month-old specific pathogen free (SPF) kittens were divided into three groups (six per group): an infected untreated group, an uninfected untreated control group, and an infected treated group (topical moxidectin and imidacloprid, Advantage Multi for Cats, Bayer Healthcare LLC). Six 2- to 3-year-old adult multiparous female SPF cats were an infected untreated adult group. The cats were evaluated by serial CBCs, bronchial-alveolar lavage (BAL), fecal examinations, thoracic radiographs, and thoracic computed tomography (CT) scans and were euthanized 65 days after the initial infection. Adult T. cati were recovered in infected untreated kittens (5/6) and infected untreated adults (5/6) in numbers consistent with natural infections. Eggs were identified in the feces of most but not all cats with adult worm infections. No adult worms were identified in the uninfected controls or the infected treated group. All cats in the infected groups, including treated cats and untreated cats without adult worms, had lung pathology based on evaluation of radiography, CT scans, and histopathology. The infected cats demonstrated a transient peripheral eosinophilia and marked eosinophilic BAL cytology, but normal bronchial reactivity based on in vivo CT and in vitro ring studies. Lung lesions initially identified by CT on day 11 were progressive. Thoracic radiographs in infected cats had a diffuse bronchial-interstitial pattern and enlarged pulmonary arteries. Pulmonary arterial, bronchial, and interstitial disease were prominent histological findings. Infected treated cats had a subtle attenuation but not prevention of lung disease compared to infected cats. Significant lung disease in kittens and adult cats is associated with the early arrival of T. cati larvae in the lungs and is independent of the development of adult worms in the intestine. These data suggest that while the medical prevention of the development of adult parasites after oral exposure to T. cati is obviously beneficial, this practice even with good client compliance will not prevent the development of lung disease which can alter clinical diagnostic methods.

HMW adiponectin is measurable via ELISA, and concentration is negatively correlated with BCS and serum insulin activity in horses. A greater understanding of the role of adiponectin in equine metabolism will provide insight into the pathophysiology of metabolic disease conditions.