Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na ⁺ -K ⁺ pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of β ₁ , but not α ₁ , subunits in rabbit ventricular myocytes at baseline. β ₁ Subunit glutathionylation was increased by peroxynitrite (ONOO ⁻ ), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased α ₁ /β ₁ subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with β ₁ subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na ⁺ -K ⁺ pump current ( I _p ). Cysteine (Cys46) of the β ₁ subunit was the likely candidate for glutathionylation. We expressed Na ⁺ -K ⁺ pump α ₁ subunits with wild-type or Cys46-mutated β ₁ subunits in Xenopus oocytes. ONOO ⁻ induced glutathionylation of β ₁ subunit and a decrease in Na ⁺ -K ⁺ pump turnover number. This was eliminated by mutation of Cys46. ONOO ⁻ also induced glutathionylation of the Na ⁺ -K ⁺ ATPase β ₁ subunit from pig kidney. This was associated with a ≈2-fold decrease in the rate-limiting E ₂ →E ₁ conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na ⁺ -K ⁺ pump occurs via glutathionylation of its β ₁ subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na ⁺ levels.

Defining the gonadal effects of FSH distinct from those of LH remains difficult. We have characterized and compared the level of Sertoli and germ cell development in three mouse models recently created to isolate FSH activity from LH effects. Two models used LH-deficient hypogonadal (hpg) mice to selectively study either pituitary-independent transgenic (tg) FSH or ligand-independent activated tg FSH receptor (FSHR(+)) expression, and the third model used LH receptor (LHR)-deficient mice to isolate and examine endogenous mouse FSH effects. Stereological evaluation revealed tg-FSH or tg-FSHR(+) activity significantly increased total Sertoli cell numbers per testis in both hpg models relative to control hpg testes. Furthermore, tg-FSH dose-dependently restored hpg Sertoli cells to wild-type (wt) (non-hpg) levels, and LHR-/- testes also exhibited wt Sertoli numbers. Spermatogonial proliferation and meiotic development were enhanced by tg-FSHR(+) or tg-FSH. Despite producing normal Sertoli numbers, isolated tg-FSH activity only increased total spermatogonia and spermatocyte populations to 57 and 44% of wt, which was comparable to spermatogonia and spermatocyte numbers observed in LHR-null testes (45 and 34% of wt). Selective FSH activity initiated round spermatid formation in all three models. However, elongated spermatid formation was detected in tg-FSH and tg-FSHR(+) hpg testes but not in LHR-/- testes, which may reflect even lower intratesticular testosterone levels in LHR-null compared with hpg testes. FSH increased round and elongated spermatid numbers in hpg testes to 16 and 6% of wt without altering intratesticular testosterone levels, but failed to produce spermatozoa demonstrating the inability of FSH to complete spermatogenesis. These findings revealed that full Sertoli cell proliferation can be accomplished by FSH activity without LH requirement, and although postnatal mitotic and meiotic germ cell development can be promoted by FSH alone, LH-mediated effects remain a critical determinant for initiating the full complement of germ cells and final stages of postmeiotic development.

Statins are a class of drugs used to lower low-density lipoprotein cholesterol and are amongst the most prescribed medications worldwide. Most statins work as a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), but statin intolerance from pleiotropic effects have been proposed to arise from non-specific binding due to poor enzyme-ligand sensitivity. Yet, research into the physicochemical properties of statins, and their interactions with off-target sites, has not progressed much over the past few decades. Here, we present a concise perspective on the role of statins in lowering serum cholesterol levels, and how their reported interactions with phospholipid membranes offer a crucial insight into the mechanism of some of the more commonly observed pleiotropic effects of statin administration. Lipophilicity, which governs hepatoselectivity, is directly related to the molecular structure of statins, which dictates interaction with and transport through membranes. The structure of statins is therefore a clinically important consideration in the treatment of hypercholesterolaemia. This review integrates the recent biophysical studies of statins with the literature on the physiological effects and provides new insights into the mechanistic cause of statin pleiotropy, and prospective means of understanding the cholesterol-independent effects of statins.

Background—: Inhibition of L-type Ca²⁺current contributes to negative inotropy of β₃adrenergic receptor (β₃AR) activation, but effects on other determinants of excitation-contraction coupling are not known. Of these, the Na⁺-K⁺pump is of particular interest because of adverse effects attributed to high cardiac myocyte Na⁺levels and upregulation of the β₃AR in heart failure.Methods and Results—: We voltage clamped rabbit ventricular myocytes and identified electrogenic Na⁺-K⁺pump current (I_p) as the shift in holding current induced by ouabain. The synthetic β₃AR agonists BRL37344 and CL316,243 and the natural agonist norepinephrine increasedI_p. Pump stimulation was insensitive to the β₁/β₂AR antagonist nadolol and the protein kinase A inhibitor H-89 but sensitive to the β₃AR antagonist L-748,337. Blockade of nitric oxide synthase abolished pump stimulation and an increase in fluorescence of myocytes loaded with a nitric oxide–sensitive dye. Exposure of myocytes to β₃AR agonists decreased β₁Na⁺-K⁺pump subunit glutathionylation, an oxidative modification that causes pump inhibition. The in vivo relevance of this was indicated by an increase in myocardial β₁pump subunit glutathionylation with elimination of β₃AR–mediated signaling in β₃AR^−/−mice. The in vivo effect of BRL37344 on contractility of the nonfailing and failing heart in sheep was consistent with a beneficial effect of Na⁺-K⁺pump stimulation in heart failure.Conclusions—: The β₃AR mediates decreased β₁subunit glutathionylation and Na⁺-K⁺pump stimulation in the heart. Upregulation of the receptor in heart failure may be a beneficial mechanism that facilitates the export of excess Na⁺.

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

The sarcolemmal Na⁺-K⁺ pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to ε-protein kinase C (εPKC), previously implicated in ANG II-induced Na⁺-K⁺ pump inhibition. A role for εPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of εPKC with the receptor for the activated kinase and with the cytosolic p47 ^phox subunit of NADPH oxidase. ANG II decreased electrogenic Na⁺-K⁺ pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by εPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na⁺-K⁺ pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The α₁-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22 ^phox and cytosolic p47 ^phox NADPH oxidase subunits at baseline. ANG II had no effect on α₁/caveolin 3 or α₁/p22 ^phox interaction, but it increased α₁/p47 ^phox coimmunoprecipitation. We conclude that ANG II inhibits the Na⁺-K⁺ pump via PKC-dependent NADPH oxidase activation.