Abstract

To better understand the molecular basis for increased atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) expression during overload-induced cardiac hypertrophy, we studied the induction of the genes in primary myocardial cells by the alpha 1-adrenergic agonist, phenylephrine (PE), a potent hypertrophic agent. PE augmented the transcription of both genes to similar extents, although the time course of mRNA accumulation differed. Increases in ANF mRNA were evident only after 6-8 h of PE exposure, when transcript levels were 2-4-fold over control. However, similar increases in BNP mRNA were observed as soon as 1 h of PE exposure. Moreover, while ANF mRNA levels continued to increase through 24 h of PE treatment, maximal levels of BNP mRNA (8-10-fold over control) were observed at 4 h, after which transcript levels declined to about 3-fold over control. The early induction of the BNP mRNA by PE was independent of protein synthesis, whereas the late induction of both genes required protein synthesis. Interestingly, the early BNP induction was only partially blocked by the transcription inhibitor, actinomycin D, indicating that, in part, the inductive effects of PE might be the result of transcript stabilization. Indeed, the BNP transcript, which was shown to possess a half-life of less than 1 h in control cells, was stabilized by the addition of PE, while the ANF transcript possessed a half-life of at least 24 h under all conditions. These data indicate that the induction of BNP by alpha 1-adrenergic agonists has characteristics of both a primary and secondary response gene, while ANF is a typical secondary response gene. Moreover, alpha 1-adrenergic stimulation enhances BNP expression through both transcriptional activation and transcript stabilization, while ANF expression is enhanced primarily transcriptionally.

Highlights

  • To better understand the molecular basis for in- known as ANF‘ and BNP, respectively, are expressed mostly in creased atrial natriureticfactor (ANF)and brain natri- the heartand released into theperipheral circulation,while the uretic peptide (BNP)expression during overload-in- C-type is expressed primarily in the brain where it probably duced cardiac hypertrophy, we studied the induction of serves as a neuromodulator,never entering the systemic circuthe genes in primary myocardial cellsby the a,adrener- lation (Komatsu et al, 1991). the relative hemodygic agonist, phenylephrine (PE), a potent hypertrophic namic roles of the cardiac-derived peptides are presently unagent

  • PE was independent of protein synthesis, whereas the Using primary myocardial cellcultures a greatdeal has been late induction of both genes required protein synthesis. learned about the regulation of ANFexpression in the heartI.t

  • Transient induction profile of some PRGs, we determined the BNP induction occurs within 1h after agonist exposure,attains rate at which the BNPmRNA accumulated in myocardial cells a maximum within several hours, and declines thereafter to in response to PE

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Summary

MATERIALS AND METHODS

Myocardial Cell Cultures a n d Dansfections-Neonatal rat ventricular myocardial cultures (>98% cardiac myocytes) were prepared as describedpreviously(Seietal., 1991; Glembotski et al, 1993). 2-fold induction of luciferase withthe BNP construct, and b6y same time PE was added, there was no production of luciferase during h, PE-induced luciferase expression wams aximal for both constructs, showingan approximately 4-5-fold increase in in trana 6- or 24-hincubation.when PE was removed from, and/or AcDt or DRB added to transfected cells, luciferase levels were shown to decline t o control within 2 h (luciferase transcript and protein degrade scription over control (Fig. 2); transcriptional activation re- rapidly). It is apparent scriptionally inducing BNP and ANF, that PE inaddition to tranalso stabilizes the similar when Act D was added when PE was removed, BNP transcript.

Time Course of ANF and BNP mRNA Induction by PE
DISCUSSION
Findings
None Act CHX
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