Use of equine embryo -derived mesenchymal stromal cells and their extracellular vesicles as a treatment for persistent breeding-induced endometritis in susceptible mares.

Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development may improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis, and early gestational tissue provides a unique source of multipotent stem cells for creating MSCs. Extracellular vesicles (EVs) are mediators of cell communication produced by many different cell types. This study utilized embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in two groups: a) PBIE-susceptible mares challenged with pooled dead sperm (n=5); and b) client-owned mares diagnosed as susceptible to PBIE (n=37 mares and 40 estrous cycles). Mares pre-treated with intrauterine EDMSCs or their EVs resulted in a significant reduction in the accumulation of intrauterine fluid post-breeding. Nine of 19 (47 %) mares treated with EDMSCs prior to natural breeding and 13 of 20 (65 %) mares treated with EDMSC derived EVs were pregnant after the first cycle and 12 of 18 (67 %) mares treated with EDMSCs, and 15 of 19 (79 %) mares treated with EVs conceived by the end of the breeding season. These preliminary clinical studies are the first reports of the use of EDMSCs or their EVs as a potential intrauterine therapy for the management of PBIE susceptible mares.

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity.

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (alpha1beta1-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol Pi/mol of alpha-subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P(i) into the alpha-subunit and the loss of activity of the Na,K-ATPase.

Phosphorylation of H,K-ATPase alpha-subunit in microsomes from rabbit gastric mucosa by cAMP-dependent protein kinase.

A 100-kDa protein that is a main component of the microsomal fraction from rabbit gastric mucosa is phosphorylated by cAMP-dependent protein kinase (PKA) in the presence of 0.2% Triton X-100. Microsomes from rabbit gastric mucosa possess activity of H,K-ATPase but not activity of Na,K-ATPase. Incubation of microsomes with 5 microM fluorescein 5'-isothiocyanate (FITC) results in both an inhibition of H,K-ATPase and labeling of a protein with an electrophoretic mobility corresponding to the mobility of the protein phosphorylated by PKA. The data suggest that the alpha-subunit of H,K-ATPase can be a potential target for PKA phosphorylation.

Mechanism of inhibition of E1-E2 ATPases by melittin.

The inhibition of Na,K-ATPase from duck salt gland and Ca-ATPase from rabbit skeletal muscle sarcoplasmic reticulum by melittin, a 26-residue peptide from bee venom, was studied. Melittin irreversibly inhibits both enzymes. At melittin/ATPase molar ratio (30-50):1, the time dependence of the inhibition is described by the sum of two exponential curves. At pH 7.0, the fast phase of the inhibition provides for about 50% of total loss of activity with pseudo-first order rate constants of 1.52 +/- 0.17 and 1.20 +/- 0.21 min-1 for Na,K- and Ca-ATPase, respectively. The corresponding pseudo-first order rate constants for the slow phase were 0.12 +/- 0.02 and 0.09 +/- 0.02 min-1. The inhibition of both enzymes by melittin depends upon pH; the inhibition increases when the pH is increased from 6.0 to 8.5. The enhancement of the inhibition concomitant to increase in pH is mainly due to an increase in the rate constant of the fast phase. ATP protects both enzymes from the inhibition by melittin; however, the character of protection is different for Ca-versus Na,K-ATPase. The protection of Ca-ATPase activity by ATP is due to an increase in melittin-insensitive activity. The protective effect of ATP on Na,K-ATPase is due to a decrease in the rate constant of fast phase as well as an increase in melittin-insensitive activity. The data suggest that the inhibition of Ca- and Na,K-ATPases by melittin results from the interaction of the peptide with two different sites. One of the sites may be located on the catalytic subunit of the enzymes, the other can be related to the lipid bilayer of the membrane.