Specific inhibition of cardiac and skeletal muscle sarcoplasmic reticulum Ca2+ pumps by H-89

Lahouratate, Philippe; Guibert, J.; Camelin, Jean-Claude; Bertrand, Ingrid

doi:10.1016/s0006-2952(97)00320-1

Cited by 22 publications

(16 citation statements)

References 38 publications

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“…Comparison with data from previous studies An inhibitory effect of H-89 on SR Ca 2+ uptake has been reported previously by Lahouratate et al [19] who showed that H-89 inhibits Ca 2+ uptake in canine cardiac and rabbit skeletal SR vesicles at similar concentrations to those used in the present study. This was also independent of changes in the phosphorylation of PLB, because A1 (a monoclonal antibody against PLB) did not inhibit the effect of H-89 on SR Ca 2+ uptake.…”

Section: Discussionsupporting

confidence: 89%

Effects of the protein kinase A inhibitor H-89 on Ca 2+ regulation in isolated ferret ventricular myocytes

Hussain

Drago

Bhogal

et al. 1999

Pfl�gers Archiv European Journal of Physiology

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We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca2+ regulation in Fura-2-loaded ferret myocytes. H-89 (10 micromol/l) decreased the amplitude of the Fura-2 transient to 28. 2+/-4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca2+ to diastolic levels: t1/2 increased from 311+/-35 ms to 547+/-43 ms (P<0.001, n=7). Reduced Ca2+ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca2+ content, as assessed with caffeine. The apparent slowing of the SR Ca2+-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca2+ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca2+-ATPase. In electrophysiological experiments, 5.0 micromol/l H-89 decreased the L-type Ca2+ current (ICa) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca2+ ]i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of beta-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca2+-ATPase.

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Section: Discussionsupporting

confidence: 89%

Effects of the protein kinase A inhibitor H-89 on Ca 2+ regulation in isolated ferret ventricular myocytes

Hussain

Drago

Bhogal

et al. 1999

Pfl�gers Archiv European Journal of Physiology

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“…8, C and D). Consistent with observations in muscle cells (42) and lymphocytes (43), H-89 itself significantly increased basal levels of [Ca 2ϩ ] i in SK-N-SH cells (Fig. 8E).…”

Section: Adm Modulates Intracellular Calcium Mobilization In Sk-n-sh supporting

confidence: 90%

Adrenomedullin Stimulates Nitric Oxide Release from SK-N-SH Human Neuroblastoma Cells by Modulating Intracellular Calcium Mobilization

Krukoff

2005

Endocrinology

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We used SK-N-SH human neuroblastoma cells to test the hypothesis that adrenomedullin (ADM), a multifunctional neuropeptide, stimulates nitric oxide (NO) release by modulating intracellular free calcium concentration ([Ca2+]i) in neuron-like cells. We used a nitrite assay to demonstrate that ADM (10 pM to 100 nM) stimulated NO release from the cells, with a maximal response observed with 1 nM at 30 min. This response was blocked by 1 nM ADM(22-52), an ADM receptor antagonist or 2 microM vinyl-L-NIO, a neuronal NO synthase inhibitor. In addition, 5 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular calcium chelator, eliminated the ADM-induced NO release. Similar results were observed when the cells were incubated in calcium-free medium or when L-type calcium channels were inhibited with 5 microM nifedipine or 10 microM nitrendipine. Depletion of calcium stores in the endoplasmic reticulum (ER) with 1 microM cyclopiazonic acid or 150 nM thapsigargin, or inhibition of ryanodine-sensitive receptors in the ER with 10 microM ryanodine attenuated the ADM-induced NO release. NO responses to ADM were mimicked by 1 mM dibutyryl cAMP, a cAMP analog, and were abrogated by 5 microM H-89, a protein kinase A inhibitor. Furthermore, Fluo-4 fluorescence-activated cell sorter analysis showed that ADM (1 nM) significantly increased [Ca2+]i at 30 min. This response was blocked by nifedipine (5 microM) or H-89 (5 microM) and was reduced by ryanodine (10 microM). These results suggest that ADM stimulates calcium influx through L-type calcium channels and ryanodine-sensitive calcium release from the ER, probably via cAMP-protein kinase A-dependent mechanisms. These elevations in [Ca2+)]i cause activation of neuronal NO synthase and NO release.

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“…This response is probably caused by the nonspecific effects of H-89 rather than its anti-PKA property because another PKA inhibitor, Rp-cAMP, has no effect on basal [Ca 2ϩ ] i . Indeed, H-89-stimulated Ca 2ϩ accumulation has been reported to occur in muscle cells (Lahouratate et al, 1997), lymphocytes (de la Rosa et al, 2001), and neuroblastoma cells (Xu and Krukoff, 2005) and is presumably caused by nonspecific effects of H-89 on the endoplasmic reticulumassociated Ca 2ϩ ATPase (de la Rosa et al, 2001). H-89 itself also decreases nNOS phosphorylation in the resting state, consistent with previous studies suggesting that PKA contributes to the constitutive phosphorylation of nNOS (Bredt et al, 1992;Dinerman et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Adrenomedullin Stimulates Nitric Oxide Production from Primary Rat Hypothalamic Neurons: Roles of Calcium and Phosphatases

Krukoff²

2007

Mol Pharmacol

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Adrenomedullin (ADM) in the brain plays important roles in the maintenance of homeostasis. Although in vivo evidence has suggested that nitric oxide (NO) mediates ADM's effects in the brain, mechanisms for ADM stimulation of NO production in neurons have not been identified. In the present study, primary hypothalamic neurons were used to characterize ADM-induced NO production and to study the underlying mechanisms. Using Calcium Orange/4-amino-5-methylamino-2Ј,7Ј-difluorofluorescein fluorescence live cell imaging, we found that ADM (1 or 10 nM, 5 min) significantly elevated [Ca 2ϩ ] i and NO production in a concentration-dependent manner. Ca 2ϩ and NO responses induced by 10 nM ADM were abolished by pretreatment with 50 M 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), an intracellular Ca 2ϩ chelator, or protein kinase A (PKA) inhibitors 5 M N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and 50 M Rp-cAMP. Furthermore, the ADM-induced NO production was significantly attenuated by a protein phosphatase 1/2A inhibitor, okadaic acid (OA; 0.1 M), or calcineurin inhibitors, tacrolimus (FK506) (1 M) and cyclosporin A (CsA; 0.1 M). Using Western blotting, we found that ADM significantly decreased phosphorylation of neuronal nitric-oxide synthase (nNOS) at serine 847. This dephosphorylation was inhibited by 0.1 M OA, 1 M FK506, 0.1 M CsA, or 5 M H-89, and attenuated by 50 M BAPTA-AM. These results suggest that, in hypothalamic neurons, ADM elevates [Ca 2ϩ ] i via PKA-associated mechanisms. The PKA/Ca 2ϩ cascade leads to protein phosphatase (PP) 1/PP2A-and calcineurin-mediated dephosphorylation of nNOS. We hypothesize that the Ca 2ϩ increase and nNOS dephosphorylation contribute to activation of nNOS and production of NO in hypothalamic neurons.

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Specific inhibition of cardiac and skeletal muscle sarcoplasmic reticulum Ca2+ pumps by H-89

Cited by 22 publications

References 38 publications

Effects of the protein kinase A inhibitor H-89 on Ca 2+ regulation in isolated ferret ventricular myocytes

Effects of the protein kinase A inhibitor H-89 on Ca 2+ regulation in isolated ferret ventricular myocytes

Adrenomedullin Stimulates Nitric Oxide Release from SK-N-SH Human Neuroblastoma Cells by Modulating Intracellular Calcium Mobilization

Adrenomedullin Stimulates Nitric Oxide Production from Primary Rat Hypothalamic Neurons: Roles of Calcium and Phosphatases

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