engleski

EFFECT OF BRPP ON NEONATAL RAT CARDIOMYOCYTE CULTURE DURING IN VITRO NOREPINEPHRINE AND ISOPROTENEROL STIMULATION

Myocardial sympathetic activity is increased in heart failure, and norepinephrine (NE)- stimulate apoptosis of cardiomyocytes through α - and β -adrenergic pathway. Isoprotenerol (ISO), as well as NE, induce apoptosis in neonatal rat cardiomyocyte (NRC) culture. Apoptosis has been demonstrated to occur in the myocardium in a variety of pathological situations. The number of apoptotic myocytes is increased in myocardium obtained from patients with end-stage heart failure and myocardial infarction and in myocardium from experimental models of myocardial hypertrophy and failure, including the aortic-banded rat, the spontaneously hypertensive rat, rats with myocardial infarction, and dogs with pacing-induced failure. Experiments with cultured cardiac myocytes have demonstrated that apoptosis can be stimulated in vitro by several endogenous peptides that are increased in the hypertrophied or failing myocardium, including TNF-α , angiotensin II and atrial natriuretic peptide, as well as with ISO and NE. BRPP (brain polipeptide) has shown, so far, positive effect on growth rate in several cell-lines aplications (CHO, BHK, ...) 1-10. MATERIAL AND METHODS Myocardial Isolation and Culture Primary ventricular cardiac myocytes were prepared from 6-8 old Fisher rats (hearts were removed, ventricles pooled and ventricular cells isolated with Worthington Neonatal Cardiomyocytes Isolation Protocol). Cells were plated to 24-well culture plates (Falcon) at density of 1, 25x105 cells/cm2 in DMEM with 10% FBS. NRC were maintained at 37oC in humidifier air with 5% CO2. After 24 hours cells were treated with NE (1 and 10 mcmol/L), ISO (1 and 10 mcmol/L) and BRPP, as well as with NE (1 and 10 mcmol/L)+BRPP and ISO (1 and 10 mcmol/L)+BRPP. 48 hours after, treated cells were harvested and counted. Confocal microscopy visualisation (α -actin staining) was performed. Isolation of BRPP The swine brain (1 kg) has been grounded in a mixer in 2 l of 0.15 M ammonium sulfate solution (pH 4.5). The mixture was stirred at 4 0C for 2 h and then centrifuged at 10 000 x g for 30 min. The pH of supernatant was adjusted to 6.5 by adding 1 M sodium hydroxide. Afterward, the supernatant was precipitated with 200 g l-1 ammonium sulfate under magnetic agitation at 4 0C for 30 min and this solution was then centrifuged at 10 000 x g for 20 min. The precipitate was discarded and supernatant was treated again with 250 g l-1 ammonium sulfate. After centrifugation, the pellet was re-suspended in 0.1 M sodium phosphate buffer (pH 6.0) and dialyzed (cellulose membrane, retention  2000 Da, Sigma) against the water overnight. The solution was then centrifuged at 15 000 x g for 10 min to remove any precipitate. The protein concentration of the extract was measured according to the method of Lowry with bovine serum albumin as standard. RESULTS NRC treated only with BRPP had significantly higher count compared to control group (p=0, 002), NE 10 (p=0, 003) and 1 mcmol/L (p=0, 001) and ISO 10 (p=0, 002) and 1 mcmol/L (p=0, 002) (figure 1). NRC groups with NE treatment with BRPP had significantly higher count compared to BRPP negative NE positive in lower concentration groups (NE 10 + BRPP – N.S. ; NE 1 mcmol/L/BRPP + p=0.001) (figure 2). NRC groups with ISO treatment with BRPP had significantly higher count in both concentration groups (ISO 10+BRPP – p=0.008 ; ISO 1+BRPP – p=0.000).

NEONATAL RAT CARDIOMYOCYTE; ISOPROTENEROL STIMULATION; NOREPINEPHRINE STIMULATION

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