Rick J. Schiebinger

Androgen Receptors Mediate Hypertrophy in Cardiac Myocytes

BACKGROUND The role of androgens in producing cardiac hypertrophy by direct action on cardiac myocytes is uncertain. Accordingly, we tested the hypothesis that cardiac myocytes in adult men and women express an androgen receptor gene and that myocytes respond to androgens by a hypertrophic response. METHODS AND RESULTS We used reverse transcription-polymerase chain reaction methods to demonstrate androgen receptor transcripts in multiple tissues and [3H]phenylalanine incorporation and atrial natriuretic peptide secretion as markers of hypertrophy in cultured rat myocytes. Messenger RNA encoding androgen receptors was detected in myocytes of male and female adult rats, neonatal rat myocytes, rat heart, dog heart, and infant and adult human heart. Both testosterone and dihydrotestosterone produced a robust receptor-specific hypertrophic response in myocytes, determined by indices of protein synthesis and atrial natriuretic peptide secretion. CONCLUSIONS Androgen receptors are present in cardiac myocytes from multiple species, including normal men and women, in a context that permits androgens to modulate the cardiac phenotype and produce hypertrophy by direct, receptor-specific mechanisms. There are clinical implications for therapeutic or illicit use of androgens in humans.

Angiotensin II-induced hypertrophy of adult rat cardiomyocytes is blocked by nitric oxide

The aim of the present study was to test the hypothesis that bradykinin-stimulated release of nitric oxide (NO) and/or prostacyclin from endothelium blocks myocyte hypertrophy in vitro. Angiotensin II increased [3H]phenylalanine incorporation by 21 ± 2% in myocytes cocultured with endothelial cells; this was abolished by bradykinin in the presence of endothelial cells. Bradykinin increased cytosolic concentrations of cGMP by 29 ± 4% in myocytes cocultured with endothelial cells. This was abolished by inhibition of NO synthase and by a cyclooxygenase inhibitor. Angiotensin II also increased [3H]phenylalanine incorporation by 28 ± 3% in myocytes cultured in the absence of endothelial cells. This effect of angiotensin II in monoculture was abolished by donors of NO but not by bradykinin. Neither the stable analog of prostacyclin (iloprost) nor the prostacyclin second messanger analog 8-bromo-cAMP (8-BrcAMP) blocked the effect of angiotensin II. Furthermore, 8-BrcAMP and iloprost individually increased [3H]phenylalanine incorporation. The antihypertrophic effects of bradykinin are critically dependent on endothelium-derived NO.The aim of the present study was to test the hypothesis that bradykinin-stimulated release of nitric oxide (NO) and/or prostacyclin from endothelium blocks myocyte hypertrophy in vitro. Angiotensin II increased [3H]phenylalanine incorporation by 21 +/- 2% in myocytes cocultured with endothelial cells; this was abolished by bradykinin in the presence of endothelial cells. Bradykinin increased cytosolic concentrations of cGMP by 29 +/- 4% in myocytes cocultured with endothelial cells. This was abolished by inhibition of NO synthase and by a cyclooxygenase inhibitor. Angiotensin II also increased [3H]phenylalanine incorporation by 28 +/- 3% in myocytes cultured in the absence of endothelial cells. This effect of angiotensin II in monoculture was abolished by donors of NO but not by bradykinin. Neither the stable analog of prostacyclin (iloprost) nor the prostacyclin second messanger analog 8-bromo-cAMP (8-BrcAMP) blocked the effect of angiotensin II. Furthermore, 8-BrcAMP and iloprost individually increased [3H]phenylalanine incorporation. The antihypertrophic effects of bradykinin are critically dependent on endothelium-derived NO.

Bradykinin Blocks Angiotensin II–Induced Hypertrophy in the Presence of Endothelial Cells

Angiotensin-converting enzyme inhibitors block left ventricular hypertrophy in vivo. A component of this effect has been attributed to tissue accumulation of bradykinin. Little is known regarding the effect of bradykinin on cardiomyocytes. The objectives of the present study were to define the effects of bradykinin on isolated ventricular cardiomyocytes (from adult and neonatal rat hearts) and to determine the extent to which bradykinin blocks hypertrophy in vitro. Bradykinin was found to be a hypertrophic agonist, as defined by increased protein synthesis and atrial natriuretic peptide secretion and expression. Bradykinin (10 micromol/L) increased [3H]phenylalanine incorporation by 23+/-3% in adult and by 36+/-10% in neonatal cardiomyocytes. Constitutive atrial natriuretic peptide secretion by neonatal myocytes was increased 357+/-103%. All effects of bradykinin were abolished by the B2-kinin receptor antagonist Hoe 140. These increases were similar in magnitude to those observed with phenylephrine (20 micromol/L) and angiotensin II (1 micromol/L). However, in cardiomyocytes cocultured with endothelial cells, bradykinin did not increase protein synthesis. Angiotensin II increased [3H]phenylalanine incorporation by 24+/-3% in adult cardiomyocytes in monoculture and by 22+/-2% in adult rat cardiomyocytes cocultured with endothelial cells. Bradykinin abolished this angiotensin II-induced hypertrophy in myocytes cultured with endothelial cells but not in myocytes studied in the absence of endothelial cells. In conclusion, bradykinin has a direct hypertrophic effect on ventricular myocytes. The presence of endothelial cells is required for the antihypertrophic effects of bradykinin. The results suggest that the increase in local concentration of bradykinin associated with angiotensin-converting enzyme inhibition is an important mechanism by which hypertrophy can be blocked. Manifestation of this mechanism appears to require bradykinin-stimulated release of paracrine factor(s) from endothelial cells, which are also able to block the hypertrophic effects of Ang II.

Calcium dependency of frequency-stimulated atrial natriuretic peptide secretion.

In this study we examined the mechanism whereby atrial natriuretic peptide secretion is increased when the frequency of contraction is raised from 2 to 5 Hz. We tested the hypothesis that calcium plays a significant role in the frequency-stimulated response. Using superfused rat left atria, we found that lowering the superfusate calcium concentration from 1.8 to 0.2 mmol/L abolished the frequency-stimulated atrial natriuretic peptide secretory response. Superfusion with ryanodine (1 mumol/L), an inhibitor of sarcoplasmic reticulum calcium release, resulted in a minimal inhibitory effect. Superfusion with 50 mumol/L nitrendipine or 10 mumol/L diltiazem inhibited the frequency-stimulated response by 46% to 48%. The lack of total inhibition suggested that an additional mechanism of calcium influx was involved, namely, inward calcium movement carried by Na(+)-Ca2+ exchange. As intracellular sodium has been reported to rise with an increase in beat frequency, a fall in the sodium gradient would favor inward calcium movement by Na(+)-Ca2+ exchange. Because we could not directly assess the role of Na(+)-Ca2+ exchange in this experimental paradigm, we examined the effect of lowering the transmembrane sodium gradient on atrial natriuretic peptide secretion by superfusion with the sodium channel activator veratridine or the sodium ionophore monensin. Superfusion with 1 mumol/L veratridine increased atrial natriuretic peptide secretion by 2.3-fold, and 1, 5, and 10 mumol/L monensin increased secretion by 1.1-, 2.1-, and 15.7-fold, respectively. In addition, we examined the possibility that the reported rise in intracellular sodium associated with increased beat frequency was due to enhanced Na(+)-H+ antiporter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin-stimulated protein synthesis by myocytes is dependent on the MAP kinase pathway and p70S6K

Bradykinin (BK) has a direct hypertrophic effect on rat ventricular cardiomyocytes (VCM) as defined by an increase in protein synthesis and an increase in atrial natriuretic peptide mRNA and secretion. In the current study, we have examined the dependence of BK-induced protein synthesis on activation of 90-kDa ribosomal S6 kinase (p90rsk) and 70-kDa S6 kinase (p70S6K). Both of these kinases possess the ability to phosphorylate the ribosomal protein S6, which plays an important role in initiating mRNA translation. Stimulation of adult VCM with 10 μM BK increased p90rsk activity by 2.5 ± 0.3-fold and increased p70S6Kactivity by 2.0 ± 0.3-fold. p90rsk is a terminal kinase in the mitogen-activated protein (MAP) kinase pathway. Inhibition of MAP kinase kinase activation by Raf in the MAP kinase pathway with PD-098059 (25 μM) blocked BK-stimulated activation of p90rsk by 70% and unexpectedly blocked p70S6K by 72%. Rapamycin inhibited BK-stimulated p70S6Kactivity by 93% but had no effect on p90rsk activation by BK. Inhibition of the MAP kinase pathway and p70S6K with PD-098059 was paralleled by changes in protein synthesis. BK (10 μM) increased [3H]phenylalanine incorporation by 27 ± 3 and 39 ± 6% in cultured adult and neonatal VCM, respectively. Treatment with PD-098059 or rapamycin abolished the increase in protein synthesis stimulated by BK. These results suggest that 1) BK activates p70S6K and p90rsk; 2) although both p70S6K and p90rsk have the potential to phosphorylate the ribosomal S6 protein, p70S6K and not p90rsk is the predominant kinase involved in increasing protein synthesis by BK; and 3) p70S6K activation is dependent on stimulation of the MAP kinase pathway at a point distal to Raf.

Insights Into the Mechanism by Which Inhibition of Na,K-ATPase Stimulates Aldosterone Production

Inhibition of Na,K-adenosine triphosphatase (Na,K-ATPase) activity by ouabain has been shown to increase the release of aldosterone from rat glomerulosa cells, but the mechanism by which this elevation of aldosterone production occurs has not been established. Small changes in membrane potential can significantly affect aldosterone release. Consequently, inhibition of Na,K-ATPase in glomerulosa cells may stimulate aldosterone production by membrane depolarization. If so, ouabain-stimulated production should be dependent on calcium influx through voltage-gated calcium channels. It has previously been shown that ouabain induces a moderately rapid increase in cytosolic calcium in rat glomerulosa cells. Therefore, in this study, we test whether ouabain stimulates aldosterone production with a time course consistent with early membrane depolarization as suggested by the previously reported early increase in cytosolic calcium. To study the time course of aldosterone production, we developed a perfusion technique that allows an examination of the initial effects of ouabain on aldosterone production. The results show that ouabain rapidly stimulates aldosterone production. Continuous perfusion with 0.25 or 1 mmol/L ouabain induced a brisk, robust increase in aldosterone production, followed by a decrease to near baseline over 60 minutes. Ouabain-stimulated aldosterone production was dependent on the presence of extracellular calcium and calcium influx through voltage-gated calcium channels. Our results support the hypothesis that the inhibition of Na,K-ATPase in rat adrenal glomerulosa cells immediately depolarizes the membrane potential and opens voltage-gated calcium channels.

Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from ∼2 to 15 μmol Pi ⋅ mg-1 ⋅ min-1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the α-subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.

Inhibitors of tyrosine phosphatases block angiotensin II inhibition of Na+ pump

To determine how angiotensin II inhibits the Na(+) pump (Na(+), K(+)-ATPase) in rat zona glomerulosa, we selectively blocked signaling proteins that could be activated by the angiotensin AT(1) receptor and known to affect Na(+) pump activity. Inhibitors of protein kinase C [calphostin C (1 microM); staurosporine (1 microM)], phospholipase A(2) [arachidonyl triflouromethyl ketone (25 microM); quinacrine (75 microM)], diacylgycerol lipase [RHC-80267 (5 microM)], and tyrosine phosphorylation [tyrphostin 47 (100 microM)] had no effect on angiotensin II inhibition of the Na(+) pump. On the other hand, inhibitors of tyrosine phosphatases [phenylarsine oxide (5 microM) and 4-bromotetramisole oxalate (100 microM)] blocked angiotensin II inhibition, where as inhibitors of serine/threonine phosphatases [okadaic acid (1 microM) and microcystin (1.5 microM)] did not. Thus, angiotensin II inhibition of the Na(+) pump may in part be mediated by a tyrosine phosphatase.