William W. Simmons

Cytokines and Insulin Induce Cationic Amino Acid Transporter (CAT) Expression in Cardiac Myocytes REGULATION OF L-ARGININE TRANSPORT AND NO PRODUCTION BY CAT-1, CAT-2A, AND CAT-2B

Cytokine-dependent production of nitric oxide (NO) by rat cardiac myocytes is a consequence of increased expression of the inducible isoform of nitric oxide synthase (iNOS or NOS2) and, in the presence of insulin, depresses the contractile function of these cells in vivo and in vitro. Experiments reported here show that L-lysine, a competitive antagonist of L-arginine uptake, suppressed NO production (detected as nitrite accumulation) by interleukin (IL)-1β and interferon (IFN) -pretreated cardiac myocytes by 70%, demonstrating that NO production is dependent on L-arginine uptake. Cardiac myocytes constitutively exhibit a high-affinity L-arginine transport system (K = 125 μM; V = 44 pmol/2 × 105 cells/min). Following a 24-h exposure to IL-1β and IFN, arginine uptake increases (V = 167 pmol/2 × 105 cells/min) and a second low-affinity L-arginine transporter activity appears (K = 1.2 mM). To examine the molecular basis for these cytokine-induced changes in arginine transport, we examined expression of three related arginine transporters previously identified in other cell types. mRNA for the high-affinity cationic amino acid transporter-1 (CAT-1) is expressed in resting myocytes and steady-state levels increase by 10-fold following exposure to IL-1β and IFN. Only cytokine-pretreated myocytes expressed a second high-affinity L-arginine transporter, CAT-2B, as well as a low-affinity L-arginine transporter, CAT-2A. In addition, insulin, which potentiated cytokine-dependent NO production independent of any change in NOS activity, increased myocyte L-arginine uptake by 2-fold and steady-state levels of CAT-1, but not CAT-2A or CAT-2B mRNA. Thus, NO production by cardiac myocytes exposed to IL-1β plus IFN appears to be dependent on the co-induction of CAT-1, CAT-2A, and CAT-2B, while insulin independently augments L-arginine transport through CAT-1.

Induction of Nitric Oxide Synthase Activity by Cytokines in Ventricular Myocytes Is Necessary but Not Sufficient to Decrease Contractile Responsiveness to β-Adrenergic Agonists

Recent evidence has documented that increased activity of an inducible nitric oxide synthase (iNOS; type 2 NO synthase) in primary isolates of adult rat ventricular myocytes after exposure to soluble mediators in medium conditioned by lipopolysaccharide-activated macrophages is associated with a decrease in their contractile responsiveness to beta-adrenergic agonists. It remained unclear which specific inflammatory cytokines in this medium contribute to the induction of iNOS activity in myocytes and whether induction of iNOS would result in an obligatory decline in contractile function. Interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNF-alpha) were both present in the lipopolysaccharide-activated macrophage-conditioned medium. However, only IL-1 receptor antagonist and not an anti-rat TNF-alpha antiserum diminished the extent of iNOS induction in myocytes exposed to this medium and prevented a decline in contractile responsiveness to isoproterenol. When recombinant cytokines were used, IL-1 beta, TNF-alpha, and IFN-gamma each induced iNOS activity in cardiac myocytes at 24 hours. However, only the combination of IL-1 beta and IFN-gamma reproducibly caused contractile dysfunction in cardiac myocytes. Among the constituents of the defined medium routinely used for maintenance of adult rat ventricular myocytes in primary culture, it was noted that insulin (10(-7) mol/L) was required for NO production, as detected by nitrite release in cytokine-pretreated myocytes, although insulin had no effect on the extent of induction of iNOS mRNA or maximal enzyme activity in myocyte cell lysates.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequency-Dependent Activation of a Constitutive Nitric Oxide Synthase and Regulation of Contractile Function in Adult Rat Ventricular Myocytes

Cardiac myocytes have recently been shown to express a constitutive Ca(2+)-sensitive isoform of NO synthase (NOS3), although the mechanism(s) responsible for activation of NOS3 and its physiological function remain to be determined. Since the activity of NOS3 is known to be regulated in part by the intracellular Ca2+ activity ([Ca2+]i) in endothelial cells, we determined whether increasing myocyte [Ca2+]i by uniform electric field pacing was accompanied by an increase in NOS3 activity, detected as nitrite accumulation in the medium. A higher [Ca2+]i with increasing pacing frequencies was shown to be accompanied by a time-dependent accumulation of nitrite in medium that bathed adult rat ventricular myocytes stimulated at 3 Hz. Nitrite release by paced cells was significantly attenuated by treatment with either the NO synthase inhibitor nitro-L-arginine (L-NA, 1 mmol/L) or the intracellular Ca2+ chelator BAPTA-AM (20 mumol/L). Paced myocytes also exhibited a frequency- and time-dependent increase in intracellular cGMP content that could be inhibited significantly by either L-NA or the soluble guanylate cyclase inhibitor LY83583 (5 mumol/L). To determine whether the increase in NOS3 activity with pacing affected contractile function, myocytes were sequentially paced at frequencies from 0.5 to 3 Hz. Methylene blue, L-NA, and LY83583 all increased the amplitude of shortening of myocytes paced at 3 Hz. Furthermore, a significantly greater positive inotropic response to high extracellular Ca2+ (3 mmol/L) was demonstrated by myocytes pretreated with L-NA compared with control cells. These data indicate that myocyte NOS3 activity is regulated in part by [Ca2+]i, whether induced by changes in pacing frequency or [Ca2+]o, and depresses myocyte contractile responsiveness to higher stimulation frequencies.

Contractile Responsiveness of Ventricular Myocytes to Isoproterenol Is Regulated by Induction of Nitric Oxide Synthase Activityin Cardiac Microvascular EndothelialCells in Heterotypic Primary Culture

Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage-conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti-rat tumor necrosis factor-alpha (TNF-alpha) antiserum. Interferon-gamma (IFN-gamma), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1 beta. Transforming growth factor-beta (TGF-beta) decreases iNOS activity, protein content, and mRNA abundance in IL-1 beta- and IFN-gamma-pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1 beta, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37 degrees C, of myocytes in heterotypic culture with IL-1 beta-pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L NG-monomethyl-L-arginine, an inhibitor of NOS.(ABSTRACT TRUNCATED AT 250 WORDS)

The NO Pathway in Cardiovascular Regulation: Constitutive and Inducible Nitric Oxide Synthase in Cardiac Myocytes and Microvascular Endothelial Cells

Nitric oxide (NO) is a ubiquitous autocrine- and paracrine-acting signalling autacoid that, among other functions, has been shown to regulate cardiac contractile responsiveness to β-adrenergic and muscarinic cholinergic agonists. Cellular constituents of cardiac muscle, including ventricular myocytes as well as microvascular endothelial cells, have been shown to express the “endothelial constitutive” isoform of NO synthase (ecNOS or NOS III) in vivo, and both cell types also express the NO synthase isoform induced by specific inflammatory cytokines (iNOS or NOS II) in vivo and in vitro. While NO-dependent intracellular signalling in cardiac myocytes clearly involves the activation of guanylate cyclase and downstream signalling by cyclic guanosine monophosphate (cGMP), there is accumulating evidence that non-cGMP-dependent regulatory signalling events are also initiated by NO. In addition, decreased contractile responsiveness of cardiac myocytes to β-adrenergic agonists, following induction of NOS II by inflammatory cytokines, requires the presence of insulin and the coinduction of enzymes responsible for production of tetrahydrobiopterin, a NOS cofactor. Inappropriate or excessive production of NO by cardiac myocytes and by microvascular endothelial cells probably contributes to the cardiac contractile dysfunction characteristic of the systemic inflammatory response syndrome and cardiac allograft rejection.