Hirotsuka Sakai

Pressure-induced expression of heat shock protein 70 mRNA in adult rat heart is coupled both to protein kinase A-dependent and protein kinase C-dependent systems

Background Production of heat shock protein 70 (HSP70) in the heart is induced by hemodynamic stress, but its intracellular signal transduction system has not been elucidated well. Objective To investigate the hypothesis that protein kinase A (PKA)-dependent and protein kinase C (PKC)-dependent systems are involved in the pressure-induced expression of HSP70 mRNA in perfused adult rat heart. Methods Isolated tetrodotoxin-arrested Sprague–Dawley rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. Aortic pressure in rats of the pressure-overloaded group was elevated from 60 to 120 mmHg for 2–120 min. cAMP contents and rates of synthesis of protein were measured by radioimmunoassay and the incorporation of [14C]phenylalanine into total heart protein, respectively. Expression of HSP70 mRNA was determined by Northern blot analysis. Results Elevation of aortic pressure significantly increased cAMP content after 2 min of perfusion (by 41%), significantly increased rates of synthesis of protein during the second hour of perfusion (by 41%), and induced expression of HSP70 mRNA maximally after 60 min of perfusion (2.7-fold the control value). Exposure to glucagon, forskolin or 1-methyl-3-isobutylxanthine mimicked increases in these parameters caused by elevation of aortic pressure. Administration of a selective PKA inhibitor, H-89, significantly prevented induction of increases in expression of HSP70 mRNA and rates of synthesis of protein by a high pressure overload and exposure to agents that increase cAMP content. Furthermore, administration of phorbol ester induced expression of HSP70 mRNA. Administration of a PKC inhibitor, calphostin C, significantly prevented induction of increases in expression of HSP70 mRNA by a pressure overload and by exposure to phorbol ester. Conclusions These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.

cAMP-mediated c -fos expression in pressure-overloaded acceleration of protein synthesis in adult rat heart

OBJECTIVES The aim was to determine whether proto-oncogene c-fos expression and acceleration of protein synthesis by acute pressure overload to the heart were coupled with a cAMP- and protein-kinase-A-dependent system in adult rat heart. METHODS Isolated adult rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. In the pressure-overloaded group, aortic pressure was raised from 60 to 120 mmHg for the time indicated. Agents that increase cAMP were added to the perfusate at an aortic pressure of 60 mmHg. Furthermore, a selective protein kinase A inhibitor (H-89) or a selective protein kinase C inhibitor (calphostin C) was administered before the elevation of aortic pressure or the addition of the agents. cAMP content or rates of protein synthesis were measured by RIA or the incorporation of [14C]phenylalanine into total heart protein, respectively. c-fos mRNA expression was determined by Northern blot analysis. RESULTS Elevation of aortic pressure in beating hearts and arrested hearts increased cAMP content at 2 min of perfusion by 36 and 41%, induced c-fos mRNA expression at 30-60 min of perfusion by 4.8- and 2.0-fold, and accelerated rates of protein synthesis during the 2nd hour of perfusion by 39 and 41% over control levels, respectively. Glucagon, forskolin or IBMX mimicked increases in these parameters by elevated aortic pressure. H-89 prevented these changes by elevated pressure overload or exposure to forskolin or IBMX in arrested hearts. On the other hand, calphostin C prevented the pressure-induced increases in c-fos expression and protein synthesis rates in arrested hearts. CONCLUSIONS These results suggest that c-fos expression induced by acute pressure overload may be coupled with increased cAMP content and protein kinase A activity in addition to increased protein kinase C activity in adult rat heart.

Genotypic Differentiation of Intrahepatically Transplanted Hyperplastic Nodule Cells of Analbuminemic and Normal Rat Origin by Polymerase Chain Reaction

DNA fragments encompassing the region of the seven‐base‐pair deletion in the albumin gene, which is a characteristic abnormality of Nagases analbuminemic rat (NAR), were amplified by polymerase chain reaction, and we could differentiate the genotypes of normal rat, homozygous NAR and heterozygous NAR electrophoretically. This genotyping method was applied to the differentiation of hyperplastic nodules on immunostained tissue sections in the intrahepatic transplantation model. When the hyperplastic nodule cells of normal rat were transplanted to the livers of homozygous NAR, the donor cells were effectively differentiated from the host cells by the genotype.

Expression of c-fos mRNA by Acute Pressure Overload in Perfused Rat Heart

It has been demonstrated that several mechanical or pharmacological overload to the heart induces immediate early gene expression such as protooncogenes and heat shock protein genes and acceler¬ates the cardiac protein synthesis to lead to cardiac hypertrophy. Norepinephrine induces cardiac hypertrophy and specific gene expression in cultured rat cardiac myocytes via α1-adrenergic recep¬tor [1,2]. Mechanical stimuli (myocyte stretching) directly induce c-fos expression in cultured neonatal rat cardiac myocytes and this response was associated with protein kinase C activation[3,4]. In these experimental models the induction of specific protooncogene expression in response to several cardiac overload might be mediated by α1-adrenergic receptor or protein kinase C-dependent signal transduction system. On the other hand, Morgan et al[5] have reported that increases in tissue cAMP content by acute pressure overload results in acceleration of protein synthesis rates in perfused adult rat hearts and that the effects of ventricular wall stretch by acute elevation of aortic pressure to accelerate synthesis may involve a cAMP-dependent protein synthesis mechanism. Our hypothesis was that protooncogene c-fos expression enhanced by acute pressure overload in adult rat hearts would play a transducing role in the cAMP-dependent protein synthesis mechanism in addition to protein kinase C-dependent mechanism. In the present study cAMP content, c-fos mRNA expres¬sion, and rates of protein synthesis were examined in adult rat hearts that were perfused at elevated aortic pressure or exposed to hormone receptor binding (glucagon) in order to determine whether c-fos expression by acute pressure overload coupled with an increase in cAMP content and continuous¬ly accelerated rates of protein synthesis.

Stretch Induces Hypertrophic Growth Through Renin-Angiotensin System in Cultured Neonatal Rat Myocytes

Cardiac hypertrophy occurs in response to various mechanical or hormonal stimuli [1]. Recentry, Baker et al [2] and Schunkert et al [3] reported that angiotensin-converting enzyme (ACE) activity, ACE mRNA expression, angiotensinogen mRNA expression and rate of angiotensin II production in heart were increased by pressure overload. Angiotensin II (A II) has two different receptors, such as type 1 angiotensin II receptor (AT1) and type 2 angiotensin II receptor (AT2) in heart [4].

Does Angiotensin II Accelerate Protein Synthesis in Adult Rat Hearts

Cardiac hypertrophy in regulated by a number of different processes such as hemodynamic load and circulating neurohumoral factors [1]. Angiotensin effects on cardiac hypertrophy are potentially of major importance and inhibitors of the renin-angiotensin system are effective therapeautic agents [2,3,4]. However, the effects of angiotensin II to stimulate cardiac hypertrophy could have been mediated by direct and indirect actions of the peptide on cardiac tissue. We have reported that elevation of aortic pressure in perfused adult rat hearts increases cAMP content and accelerates ribosomal formation and protein synthesis [5,6]. This model was used to examine the direct effects of angiotensin II on cardiac hypertrophy. The purpose of these expriments were to: 1) determine if angiotensin II accelerated rates of protein synthesis; and 2) evaluate if angiotensin coverting enzyme (ACE) inhibitors prevented the effects of elevated aortic pressure on protein synthesis.