There are 2 main categories of angiotensin II (Ang II) receptors, Ang II type 1 (AT1) and Ang II type 2 (AT2), which coexist in cardiac muscle cell membrane. Indeed, the expression of AT1R has no obvious variation up to majority. AT2R is low-expressed in cardiac myocytes of adult rat, while the expression of AT1R and AT2R is up-regulated in senescent hearts.1 In addition, the AT1R stimulation is known to promote cardiovascular hypertrophy and fibrosis,2 whereas AT2R activation is believed to oppose AT1R-mediated effects.3 But, there are no experimental results about the interactions of signal transduction of AT1R and AT2R in the process of senescent hearts. The present study was designed to investigate the enzyme activities relevant to the interactions of signal transduction between AT1R and AT2R, and to detect whether there is any difference in the interaction in rat hearts of different age. We hope to elucidate some mechanisms underlying the interactions of signal transduction.
Ang II, PD123319 (a selective antagonist of AT2R), prazosin and tyrosine kinase assay kit were obtained from Sigma-Aldrich Inc. (USA); losartan (a selective antagonist of AT1R) was provided by Merck & Co. Inc. (USA). PepTag® Assay for non-radioactive detection of protein kinase C (PKC) system was from Promega Co. (USA). Bradford protein assay kit was purchased from Applygen Technologies Inc. (China). cPLA2 assay kit system was from Cayman Chemical Co. (USA). 125I-cGMP radioimmunoassay kit for enzymatic assay was provided by Chinese Traditional Medical College of Shanghai (China).
Male Wistar rats (purchased from Vital River Lab Animal Technology, Co. Ltd. Beijing, China) at different ages (3.5, 12, 18 and 24 months old) were used in this study. All experimental procedures and protocols conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication No.85-23, revised 1985). The rats were anesthetized with 40 mg/kg sodium pentobarbitone, i.p., and decapitated. The hearts of the rats were carefully excised and washed with modified Krebs solution (mmol/L: NaC1 118.3, KC1 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, EDTA 0.026, NaHCO3 25.0 and glucose 11.1, pH 7.40). After removal of the connective tissue, blood vessels, adherent fat, atria, and the right ventricle, the left ventricle was weighed, minced and incubated in modified Krebs solution, which was kept at 37°C and aerated with 95% O2 and 5% CO2.
Measurement of PKC activities
To investigate whether the activation of AT2R has any effects on PKC activities stimulated by the activation of AT1R, 100 mg heart fragments were perfused for 15 minutes with Ang II (1×10-8 mol/L) plus prazosin (1×10-7 mol/L) (n= 6) and 100 mg heart fragments with Ang II plus prazosin (1×10-7 mol/L) and the AT2R blocker PD122319 (5×10-7 mol/L) (n=6). The alpha1-adrenoceptor blocker prazosin was used to prevent any indirect stimulation of PKC via activation of the postsynaptic sympathetic system.4,5 The dose of PD122319 was chosen on the basis of previous studies demonstrating a selective AT2R blockade in response to Ang II.6 This period was chosen on the basis of the time of previous studies.7,8 Tissue samples after perfusion were snap-frozen in liquid nitrogen for later use. Membrane fractions from frozen tissue were prepared as described previously.7 PKC was detected by PepTag® assay for non-radioactive detection of the PKC system. One unit of kinase activity was defined as the number of nanomoles of phosphate transferred to a substrate per minute per milligram protein, and protein concentration was determined using the Bradford protein assay.
Measurement of tyrosine kinase activities
To investigate whether the activation of AT2R has any effects on tyrosine kinase activities stimulated by the activation of AT1R, additional 100 mg heart fragments were subjected to Ang II (1×10-8 mol/L) plus prazosin (1×10-7 mol/L) (n=6) and 100 mg heart fragments to Ang II plus prazosin (1×10-7 mol/L) and AT2R blocker PD123319 (5×10-7 mol/L) (n=6). Tyrosine kinase activities were determined by tyrosine kinase assay. The tyrosine kinase activity in the sample was extrapolated from the EGFR activity graph (absorbance at 492 nm vs units EGFR activity).
Measurement of cPLA2 activities
To investigate whether the activation of AT1R has any effects on cPLA2 activities stimulated by the activation of AT2R, another 100 mg heart fragments were perfused with Ang II (1×10-8 mol/L) plus prazosin (1×10-7 mol/L) (n=6), and 100 mg heart fragments with Ang II plus AT1R blocker losartan (1×10-7 mol/L) (n=6). The dose of losartan was chosen on the basis of previous studies demonstrating a selective AT1R blockade in response to Ang II.9 cPLA2 was detected by the cPLA2 assay kit system. The cPLA2 activity in the sample was extrapolated from the bee venom PLA2 activity graph (absorbance at 405 nm vs units bee venom PLA2).
Measurement of cGMP content
To investigate whether the activation of AT1R has any effects on cGMP content stimulated by the activation of AT2R, additional 100 mg heart fragments were subjected to Ang II (1×10-8 mol/L) plus prazosin (1×10-7 mol/L) (n=6) and 100 mg heart fragments to Ang II plus prazosin (1×10-7 mol/L) and AT1R blocker losartan (1×10-7 mol/L) (n=6). cGMP levels were determined by 125I-cGMP radioimmunoassay kits enzymatic assay and expressed as mol/mg protein.8
All data were expressed as means ± standard deviation (SD). Student’s paired t test was used to determine the statistical significance of differences. All the data were evaluated statistically with SPSS 11.5 software. Statistical significance was accepted at P<0.05.
Effects of AT2R activation on PKC activities induced by AT1R activation
When AT1R and AT2R were activated by Ang II compared to AT2R antagonist in parallel with Ang II, the membrane activities of PKC in the 3.5- and 12-month-old groups were not significantly different. In the 18- and 24-month-old groups, however, the membrane activities of PKC were significantly decreased (Fig. 1).
Effects of AT2R activation on tyrosine kinase activities induced by AT1R activation
Additional heart fragments were studied to examine the effect of AT2R blockade on tyrosine kinase activities in rat heart of different age. As in the first protocol, the activation of AT2R had no result in AT1R-induced tyrosine kinase activities in the 3.5-month-old group. After AT2R was activated in 12-,18- and 24-month-old rat hearts, the tyrosine kinase activities were decreased significantly in contrast to those after AT1R was activated alone (Fig. 2).
Effects of AT1R activation on cPLA2 activities induced by AT2R activation
The cPLA2 activities were significantly decreased in rat heart of different age when AT1R was activated in contrast to when AT2R was activated alone (Fig. 3).
Effects of AT1R activation on the cGMP content induced by AT2R activation
When rat heart was stimulated with Ang II, AT1R and AT2R were all activated in contrast to that just AT2R was activated by Ang II and losartan; the cGMP levels were significantly decreased in rat hearts of different age (Table).
Using a buffer-perfused heart muscle model, we directly examined the effect of AT2R blockade that modulates Ang II-stimulated PKC and tyrosine kinase activities. We also directly examined the effect of AT1R blockade on Ang II-stimulated cPLA2 activities and cGMP content in Ang II-perfused heart muscle. It is well-known that both AT1R and AT2R can be activated by Ang II, while AT2R blockade PD123319 or AT1R blockade losartan at the presence of Ang II just activates AT1R or AT2R. Thus, the data suggest the interaction between AT1R and AT2R activation.
PKC is one of the important enzymes in the signal transduction pathway of AT1R. AT1R coupled to the heterotrimer G-protein Gq activates phospholipase Cβ, which generates inositol trisphosphate and diacylglycerol (DAG) via a calcium-mediated pathway. DAG can activate PKC, which translates PKC from cytolist to membrane. Experimentally, normal hearts stimulated with Ang II demonstrated a significant increase in the membrane fraction of PKC, which was not further modified by AT2R blockade8,10 At the presence of PD123319 in hypertrophied hearts, however, Ang II significantly increased the membrane fraction of PKC compared with hypertrophied hearts with no AT2 blockade. Obviously, senescent rats characteristically developed age-related cardiac hypertrophy and fibrosis. Hence our findings were consistent with previous observations.11,12 We found that AT2R blockade boosted up the activities of heart muscle membrane fraction of PKC compared to perfusion with PD123319 during aging variation. That is to say, the AT2R activation inhibits AT1R-activated PKC during heart aging variation.
Several mechanisms have been shown to mediate the activation of non-receptor tyrosine kinases and receptor tyrosine kinases by the G-protein-coupled receptors.13-15 Ang II induced activation of AT1R stimulates extracellular signal-regulated kinase (ERK) 1/2 phosphorylation via transactivation of the receptor tyrosine kinases or non-receptor tyrosine kinases. This leads to phosphorylation of the tyrosine kinases as well as its subsequent internalization,16 which can induce the effect of promoting cell growth, migration and inflammation. In our study, the activities of tyrosine kinases were increased at the presence of AT2R blockade in the 12-month-old rat. That is to say the activation of AT2R inhibited the AT1R activation from then on because AT2R coupled to tyrosine phosphatase, which can inhibit the tyrosine kinases activites.17-19 Because Ang II binds to its two receptor subtypes AT1 and AT2 with a similar affinity,20 the cellular response will be highly dependent on the relative expression level and/or responsiveness of both receptors. Experimentally,21 the expression of the AT2R gene was lowered in adult rats but it was increased in both ventricles of senescent rats. Furthermore AT2R specific binding was increased in the aged left ventricular myocardium. AT2R had a stronger inhibition of AT1R activated cellular proliferation, and AT2R blockade reversed AT1R mediated regression of cardiovascular hypertrophy and fibrosis in senescent rats.12 Our results suggest that these effects are possibly related to AT2R coupled to tyrosine phosphatase, inhibiting the activities of AT1R tyrosine kinases.
Though the signal mechanism of AT2R is not clear, three major transduction mechanisms are responsible for AT2R signaling: (a) the activation of various protein phosphatases causing protein dephosphorylation; (b) activation of the NO/cGMP system; and (c) stimulation of phospholipase A2 with subsequent release of arachidonic acid.20,22 Our studies demonstrated that AT1R blockade causes an increase of cPLA2 activities and cGMP content stimulated by Ang II in rat hearts of different age, which suggests that AT1R activation can inhibit the activation of AT2R in all rat hearts.
Traditionally, the major effects of ATR are thought to be induced by AT1R, but recent experimental evidences suggest that in senescent hearts and some pathological conditions such as hypertension and myocardial hypertrophy, AT2R actually plays an important role.12,23 The present results suggest that AT2R and AT1R have opposing effect each other in signal transduction. Ang II acting at AT1R has well documented effects on cardiovascular structure such as the promotion of cardiovascular hypertrophy and fibrosis,3 which are believed to be opposed by AT2R stimulation.4 In addition, the expression of AT1R and AT2R is up-regulated both in hypertrophied heart23 and with increasing age,22 and the function of senescent heart is more dependent on Ang II.12 The results of the present study illustrate that AT1 and AT2 receptor subtypes have more negative crosstalk signal transduction in senescent hearts.
In conclusion, the activation of AT2R inhibits the signal mechanism of AT1R in senescent hearts, and the activation of AT1R inhibits signal mechanism of AT2R in rat hearts of different age. The beneficial or detrimental effects of the negative crosstalk signal transduction remain to be investigated. The present study has the following limitations ie the isolated-perfused heart model does not allow investigation of later effect of the negative crosstalk such as the effect on cellular proliferation, and it does not distinguish whether changes in enzyme activities or cGMP content are localized predominantly to cardiac myocytes or also to matrix cells that express Ang II receptors. However, this is the first report about the presence of this interaction in signal transduction. The onset and role of this alteration in the course of heart insenecence needs further evaluation, and a better understanding of cross regulation between the two receptor subtypes and potential mechanisms is necessary for these crosstalks of intracellular signal.
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