Tyrosine kinase and mitogen-activated protein kinase/extracellularly regulated kinase differentially regulate intracellular calcium concentration responses to angiotensin II/III and bradykinin in rat cortical thick ascending limb.

The cortical thick ascending limb (CTAL) coexpresses angiotensin (Ang) II/Ang III receptor type 1A (AT(1A)-R) and bradykinin (BK) receptor type 2 (B2-R). In several cell types, these two receptors share the same signaling pathways, although their physiological functions are often opposite. In CTAL, little is known about the intracellular transduction events leading to the final physiological response induced by these two peptides. We investigated and compared in this segment the action of Ang II/III and BK on intracellular calcium concentration ([Ca2+]i) response and metabolic CO2 production, an index of Na+ transport, by using inhibitors of protein kinase C (bisindolylmaleimide), Src tyrosine kinase (herbimycin A and PP2), and MAPK/ERK (PD98059 and UO126). Ang II/III and BK (10(-7) mol/liter) released Ca2+ from the same intracellular pools but activated different Ca2+ entry pathways. Ang II/III- or BK-induced [Ca2+]i increases were similarly potentiated by bisindolylmaleimide. Herbimycin A and PP2 decreased similarly the [Ca2+]i responses induced by Ang II/III and BK. In contrast, PD98059 and UO126 affected the effects of BK to a larger extent than those of Ang II/III. Especially, the Ca2+ influx induced by BK was more strongly inhibited than that induced by Ang II/III in the presence of both compounds. The Na+ transport was inhibited by BK and stimulated by Ang II/III. The inhibitory action of BK on Na+ transport was blocked by UO126, whereas the stimulatory response of Ang II/III was potentiated by UO126 but blocked by bisindolylmaleimide. These data suggest that the inhibitory effect of BK on Na+ transport seems to be directly mediated by an increase in Ca2+ influx dependent on MAPK/ERK pathway activation. In contrast, the stimulatory effect of Ang II/III on Na+ transport is more complex and involves PKC and MAPK/ERK pathways.

Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arterioles of rat kidney.

ANG II controls the vascular tone of pre- and postglomerular arterioles, and thereby glomerular filtration, through binding to either AT1A, AT1B, or AT2 receptors. AT1 receptors, which are coupled to intracellular Ca2+ signaling, have vasoconstricting effects, whereas AT2 receptors, whose signaling mechanism is unknown, induce vasodilatation. The angiotensin receptors have been characterized in afferent arterioles, which express the three types of receptors, but not in efferent arterioles. Two subpopulations of juxtamedullary efferent arterioles, muscular ones which terminate as vasa rectae and thin ones which terminate as peritubular capillaries, have been described. They display functional heterogeneity with regard to the ANG II response. To evaluate whether these differences are associated with differential expression of ANG II receptors, we examined the expression pattern of AT1A, AT1B, and AT2 receptor mRNAs by RT-PCR in these arterioles and studied the effect of valsartan, a specific AT1-receptor antagonist. Results indicate that muscular arterioles express AT1A, AT1B, and AT2 receptors, whereas thin arterioles only express the AT1A and AT2 types, and at a much lower level. Valsartan fully inhibited ANG II-induced increases in intracellular Ca2+ in both arteriolar types, but with different kinetics. In muscular arterioles, inhibition was monoexponential, whereas it displayed a marked positive cooperativity in thin arterioles. Finally, the apparent affinity for valsartan was higher in muscular than in thin arterioles. In conclusion, this study further documents the differences between muscular and thin efferent arterioles with regard to ANG II signalization in the rat kidney.

ACE and non-ACE mediated effect of angiotensin I on intracellular calcium mobilization in rat glomerular arterioles.

Because renin and angiotensin I (ANG I) level are high in the renal circulation, the conversion of ANG I is a critical step in the regulation of glomerular hemodynamics. We studied this conversion by investigating the effect of ANG I on intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat juxtamedullary glomerular afferent and efferent arterioles (AA and EA, respectively). Two types of EA were considered, thin EA and muscular EA, terminating as peritubular capillaries and vasa rectae, respectively. In all arterioles, ANG I elicited [Ca(2+)](i) elevations. Maximal responses of 171 +/- 28 (AA), 183 +/- 7 (muscular EA), and 78 +/- 11 nM (thin EA) (n = 6), similar to those obtained with ANG II, were observed with 100 nM ANG I. The EC(50) values were 20 times higher for ANG I than for ANG II in AA (10.2 vs. 0.5) and muscular EA (6.8 vs. 0.4 nM) and 150 times higher in thin EA (15.2 vs. 0.1 nM). ANG I effect was blocked by losartan, indicating that AT(1) receptors were involved. The ANG-converting enyzme (ACE) inhibitor lisinopril inhibited the maximal response to ANG I in AA and muscular EA by 75 +/- 9% (n = 13) and 70 +/- 7% (n = 13), respectively, but had no effect in thin EA (n = 14). The serine protease inhibitor aprotinin, the chymase inhibitor chymostatin, and the cysteine protease inhibitors E64 and leupeptin had no effect on ANG I action. These data show that ANG I effects are mainly mediated by ACE in AA and muscular EA but not in thin EA. The lisinopril-insensitive response may be related to conversion by unknown enzyme(s) and/or to activation of AT(1) receptors by ANG I.

Potentiation of [Ca2+]i response to angiotensin III by cAMP in cortical thick ascending limb.

BACKGROUND: In the rat cortical thick ascending limb (CTAL), intracellular Ca2+ ([Ca2+]i) responses to angiotensin II (Ang II) and angiotensin III (Ang III) were mediated by the Ang II subtype 1A receptor (AT1A-R), whereas the arginine vasopressin (AVP)-dependent cAMP accumulation involved the vasopressin receptor type 2 (V2-R). This work was performed in CTAL to investigate the crosstalk between these two receptors by studying their transduction pathways. METHODS: The cAMP-dependent pathway was activated by 10 minutes of prestimulation with either forskolin, CTP-cAMP or AVP, and Ang II/Ang III-induced [Ca2+]i responses were assessed. RESULTS: Pretreatment with 5 micromol/L forskolin significantly enhanced the [Ca2+]i response induced by 10-7 mol/L either Ang II or Ang III. Analysis of dose-response curves to Ang III in forskolin-treated CTAL demonstrated that the maximal [Ca2+]i response was significantly increased without altering the EC50. In Ca2+-free medium, the forskolin-induced potentiation of the [Ca2+]i response to Ang III was weaker but always present, suggesting that this effect was not only due to intracellular Ca2+ release but also to extracellular Ca2+ influx. Furthermore, the fact that the forskolin-induced potentiation of the [Ca2+]i response to Ang III was blocked by 10 micromol/L H-89, a specific protein kinase A (PKA) inhibitor, indicated that this effect occurred via activation of PKA. Finally, the potentiation of the [Ca2+]i response to Ang III also was observed following pretreatment with 100 micromol/L CTP-cAMP or 10-7 mol/L AVP. CONCLUSIONS: In CTAL, there is a positive crosstalk between the adenylyl cyclase and phosphoinositide pathways mediated by V2- and AT1A-R, respectively, through activation of PKA.