The contractility index showed positive inotropic effects

The contractility index showed positive inotropic effects of apelin in both doses at min 5 (Fig. 3, Fig. 5). Although the mean arterial pressure did not recover to the baseline, the contractility index increased, the relaxation index (tau) and ±dp/dt max returned back to their baseline, and left ventricle end diastolic pressure (LVEDP) also decreased in response to apelin 60; all these changes indicated positive inotropic and lusitropic effects of apelin at min 5. The study of Perjes and colleagues in isolated rat heart showed that the effect of apelin on cardiac contractility appears at least 3min after its administration [8]. Therefore changes in these parameters observed in min 1 in the present study are consequences of the alterations in afterload (arterial blood pressure) and their related effects on myocardial performance and not the consequences of direct effect of apelin on the heart. The APJ and kappa opioid receptors (KORs) are the mediators of negative inotropic responses to apelin, because the inhibition of APJ and KORs prevents the reduction of contractility index, in response to apelin 60μg/kg (Fig. 5A). The inhibition of KORs prevents the significant reduction of +dp/dt max at min 1 in response to apelin 60μg/kg (Fig. 4C). It is suggested that KORs are the mediator for negative inotropic effects of apelin. The negative lusitropic effects of apelin are not induced through KORs, because the inhibition of KORs has no significant effect on –dp/dt max and tau (Figs. 4C and 5C). These findings suggest that these changes may also be related to the indirect effects of apelin on the heart contractility through changes in arterial blood pressure. It seems that vasodilation (pressure lowering) effects of apelin start quicker than its potentiating effects on the heart. It has also been shown that KORs are the SCH 527123 opioid receptors on the vasculature [20]. The APJ and KORs are also the mediators for positive inotropic responses to apelin, as the inhibition of these receptors prevents the increase of contractility index in response to apelin (Fig. 5B). The initial negative inotropic effect of apelin is also inhibited by F13A and nor-BNI (Fig. 5A). These findings suggest that both early (negative) and late (positive) inotropic effects of apelin are mediated by APJ and KORs, the former indirectly through blood pressure and the latter directly on the heart. Heterodimerization between KOR and APJ is one of the mechanisms suggested for the interaction between the receptors. It has been shown that GPCRs form heterodimer or oligodimers with each other and modify each other's function via physical interaction or regulation of downstream signaling pathways [21]. Results of the present study indicated that KOR and APJ form heterodimer in LV, and hypertension increases the level of heterodimerization. Increase in the expression of receptors is one of the factors that probably result in the increase of heterodimerization between the receptors. Results of the present study showed that the expression of KOR protein increased in the LV of 2K1C rats (Fig. 6B). It has been shown that, increasing the expression of bradykinin receptor increases the heterodimerization between angiotensin type 1 (ATR1) and bradykinin receptors [21]. Therefore it is possible that increase in the expression of KORs increases the heterodimerization between KORs and APJ in the heart of rats with renovascular hypertension. Further investigations are required to clear whether the increased expression of KORs and increased heterodimerization between KORs and APJ in the heart imposed to pressure load, are protective mechanisms in these conditions or play a role in the process of developing cardiac hypertrophy seen in these animals (Fig. 1). It is proposed that for more conclusive evidences of the above findings, in future studies, the dimerization of KOR and APJ to be confirmed using synthetics peptides that mimic the transmembrane domain sequences of the two receptors to attempt to disrupt the interaction.