Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness

Several examples of functional G-protein-coupled receptor heterodimers have been identified. However, it is not known whether receptor heterodimerization is involved in the pathogenesis of human disorders. Here we show that in preeclamptic hypertensive women, a significant increase in heterodimerization occurs between the AT(1)-receptor for the vasopressor angiotensin II and the B(2)-receptor for the vasodepressor bradykinin. AT(1)-B(2)-receptor heterodimerization in preeclampsia correlated with a 4-5-fold increase in B(2)-receptor protein levels. Expression of the AT(1)-B(2) heterodimer increased the responsiveness to angiotensin II and conferred resistance in AT(1)-receptors to inactivation by reactive oxygen species raised in normotensive and preeclamptic pregnancies. We suggest that AT(1)-B(2) heterodimers contribute to angiotensin II hypersensitivity in preeclampsia. Moreover, we identify preeclampsia as the first disorder associated with altered G-protein-coupled receptor heterodimerization.     

Spontaneously beating neonatal rat heart myocyte culture-a model to characterize angiotensin II at(1) receptor autoantibodies in patients with preeclampsia

This report focuses on angiotensin II AT(1) receptor autoantibodies (anti-AT(1)-AABs) in preeclamptic women. An enzyme-linked immunosorbent assay was described. Biotinylated peptide was incubated with anti-AT(1)-AABs. Streptavidin-coated magnetic particles bind the protein-autoantibody complex. Detection of anti-AT(1)-AABs was performed using anti-human IgG3 peroxidase-coupled antibody. The color reaction of tetramethylbenzidine solution was stopped by adding 0.5 M H(2)SO(4). Optical density was measured at 450 nm (620 nm reference filter). Seventy-nine percent of anti-AT(1)-AAB-positive patients (measured by bioassay) showed an increase in optical density (>145%). The same biotinylated peptide was successfully used for purification of 6/6 anti-AT(1)-AABs. Chronotropic effects of purified antibodies were registered on primary cultured neonatal rat cardiomyocytes with the computer imaging system IMAGOQUANT. Western blot of coimmunoprecipitation of angiotensin II AT(1) receptor shows one band (molecular weight >40.0 kDa) in potassium thiocyanate eluate.     

The angiotensin II AT2 receptor is an AT1 receptor antagonist

The vasopressor angiotensin II activates AT(1) and AT(2) receptors. Most of the known in vivo effects of angiotensin II are mediated by AT(1) receptors while the biological functions of AT(2) receptors are less clear. We report here that the AT(2) receptor binds directly to the AT(1) receptor and thereby antagonizes the function of the AT(1) receptor. The AT(1)-specific antagonism of the AT(2) receptor was independent of AT(2) receptor activation and signaling, and it was effective on different cells and on human myometrial biopsies with AT(1)/AT(2) receptor expression. Thus, the AT(2) receptor is the first identified example of a G-protein-coupled receptor which acts as a receptor-specific antagonist.     

Perindopril treatment promote left ventricle remodeling in patients with heart failure screened positive for autoantibodies against angiotensin II type 1 receptor

Coronary artery disease (CAD) is accountable for more than 7 million deaths each year according to the World Health Organization (WHO). In a European population 80% of patients diagnosed with CAD are overweight and 31% are obese. Physical inactivity and overweight are major risk factors in CAD, thus central strategies in secondary prevention are increased physical activity and weight loss. In a randomized controlled trial 70 participants with stable CAD, age 45–75, body mass index 28–40 kg/m2 and no diabetes are randomized (1:1) to 12 weeks of intensive exercise or weight loss both succeeded by a 40-week follow-up. The exercise protocol consist of supervised aerobic interval training (AIT) at 85-90% of VO2peak 3 times weekly for 12 weeks followed by supervised AIT twice weekly for 40 weeks. In the weight loss arm dieticians instruct the participants in a low energy diet (800–1000 kcal/day) for 12 weeks, followed by 40 weeks of weight maintenance combined with supervised AIT twice weekly. The primary endpoint of the study is change in coronary flow reserve after the first 12 weeks’ intervention. Secondary endpoints include cardiovascular, metabolic, inflammatory and anthropometric measures. The study will compare the short and long-term effects of a protocol consisting of AIT alone or a rapid weight loss followed by AIT. Additionally, it will provide new insight in mechanisms behind the benefits of exercise and weight loss. We wish to contribute to the creation of effective secondary prevention and sustainable rehabilitation strategies in the large population of overweight and obese patients diagnosed with CAD. ClinicalTrials.gov: NCT01724567     

Superimposition of potent non-peptide AT1 receptor antagonists with angiotensin II

Summary The model of angiotensin II (ANG II) developed in our laboratory using a combination of NMR, fluorescence data and molecular graphics [Matsoukas, J.M. et al., J. Biol. Chem., 269 (1994) 5303] served as a template for a systematic superimposition of potent AT₁ receptor antagonists with ANG II. The key amino acids in this model, tyrosine, phenylalanine and histidine, form a charge-relay system. The studied ANG II AT₁ receptor antagonists were found to accommodate this relay system. The proposed model offers a motivation to synthetic chemists to develop ANG II antagonists that differ from the losartan prototype structure but possess an enhanced biological profile.     

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT₁ and AT₂), defined as intracrine response. The aim of this study was to examine the presence of AT₁ and AT₂ receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT₁ through an intracrine mechanism. Subcellular distribution of AT₁ and AT₂ was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

     

Intracellular angiotensin II induces cell proliferation independent of AT1 receptor

We recently reported intracrine effects of angiotensin II (ANG II) on cardiac myocyte growth and hypertrophy that were not inhibited by the ANG II type 1 receptor (AT₁) antagonist, losartan. To further determine the role of AT₁ in intracrine effects, we studied the effect of intracellular ANG II (iANG II) on cell proliferation in native Chinese hamster ovary (CHO) cells and those stably transfected with AT₁ receptor (CHO-AT₁). CHO-AT₁, but not CHO cells, showed enhanced proliferation following exposure to extracellular ANG II (eANG II). However, when transiently transfected with an iANG II expression vector, both cell types showed significantly enhanced proliferation, compared with those transfected with a scrambled peptide. Losartan blocked eANG II-induced cell proliferation, but not that induced by iANG II. To further confirm these findings, CHO and CHO-AT₁ cells were stably transfected for iANG II expression (CHO-iA and CHO-AT₁-iA, respectively). Cells grown in serum-free medium were counted every 24 h, up to 72 h. CHO-iA and CHO-AT₁-iA cells showed a steeper growth curve compared with CHO and CHO-AT₁, respectively. These observations were confirmed by Wst-1 assay. The AT₁ receptor antagonists losartan, valsartan, telmisartan, and candesartan did not attenuate the faster growth rate of CHO-iA and CHO-AT₁-iA cells. eANG II showed an additional growth effect in CHO-AT₁-iA cells, which could be selectively blocked by losartan. These data demonstrate that intracrine ANG II can act independent of AT₁ receptors and suggest novel intracellular mechanisms of action for ANG II. renin-angiotensin system; angiotensinogen; peptide hormones; nuclear signaling; intracrine     

Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII interacts with the second extracellular loop of the AT1 receptor.     

Chronic angiotensin II AT1 receptor blockade increases cerebral cortical microvessel density

Angiotensin II is known to stimulate angiogenesis in the peripheral circulation through activation of the angiotensin II type 1 (AT1) receptor. This study investigated the effect of angiotensin receptor blockade on cerebral cortical microvessel density. Rats (6-7 wk old, n = 5-17) were instrumented with femoral arterial and venous indwelling catheters for arterial blood pressure measurement and drug administration. Rats were treated for 3 or 14 days with the AT1 receptor blocker losartan (50 mg/day in drinking water) or vehicle. Brains were sectioned and immunostained for CD31, and microvessel density was measured. Treatment with losartan for 3 or 14 days resulted in a slight decrease in mean arterial blood pressure (3 days, 92 +/- 1 mmHg; and 14 days, 99 +/- 2 mmHg) compared with vehicle (109 +/- 3 and 125 +/- 4 mmHg, respectively). A furosemide + captopril 14-day treatment group was added to control for the blood pressure change (96 +/- 3 mmHg). Microvessel density increased in groups treated with losartan for 14 days (429 +/- 13 vessels/mm2) compared with vehicle (383 +/- 11 vessels/mm2) but did not change with furosemide + captopril (364 +/- 7 vessels/mm2). Thus AT1 receptor blockade for 14 days resulted in increased cerebral microvessel density in a blood pressure-independent manner.     

Cerebroprotective effects of angiotensin II (AT 1) receptor antagonists?

The results of the Acute Candesartan Cilexetil Therapy in Stroke Survivors (ACCESS) study show that treatment with an angiotensin receptor blocker (ARB) in the acute phase of a stroke improves mortality and cardiovascular morbidity. In addition, direct comparative antihypertensive trials have demonstrated beneficial effects of ARBs in preventing stroke. These possible cerebro-protective effects of ARBs are supported by animal studies, demonstrating that stimulation of the AT2 receptor was related to a reduction in both cerebral infarct size and mortality. In the present report, we review both pathophysiological and clinical evidence for possible cerebroprotective effects of ARBs, independent of their effect on blood pressure.     

Interaction of angiotensin II with the C-terminal 300-320 fragment of the rat angiotensin II receptor AT1a monitored by NMR

Interaction between angiotensin II (Ang II) and the fragment peptide 300-320 (fCT300-320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9Lys(fCT) and 6His(ang), 10Phe(fCT) and 8Phe(ang), HN proton of 3Tyr(fCT) and Halpha of 4Tyr(ang), 5Phe(fCT)Hdelta and Halpha of 4Tyr(ang) indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300-320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an alpha-helix backbone structure to fCT300-320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment.     

Endogenous angiotensin II suppresses stretch-induced ANP secretion via AT1 receptor pathway

Angiotensin II (Ang II) is released by stretch of cardiac myocytes and has paracrine and autocrine effects on cardiac myocytes and fibroblasts. However, the direct effect of Ang II on the secretion of atrial natriuretic peptide (ANP) is unclear. The aim of the present study is to test whether Ang II affects stretch-induced ANP secretion. The isolated perfused beating atria were used from control and two-kidney one-clip hypertensive (2K1C) rats. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load caused increases in atrial contractility by 60% and in ANP secretion by 100%. Ang II suppressed stretch-induced ANP secretion and tended to increase atrial contractility whereas losartan stimulated stretch-induced ANP secretion. Neither PD123319 nor A779 had direct effect on stretch-induced ANP secretion. The suppressive effect of Ang II on stretch-induced ANP secretion was blocked by the pretreatment of losartan but not by the pretreatment of PD123319 or A779. In hypertrophied atria from 2K1C rats, stretch-induced ANP concentration attenuated and atrial contractility augmented. The response of stretch-induced ANP secretion to Ang II and losartan augmented. The expression of AT1 receptor protein and mRNA increased but AT2 and Mas receptor mRNA did not change in 2K1C rat atria. Therefore, we suggest that Ang II generated endogenously by atrial stretch suppresses stretch-induced ANP secretion through the AT1 receptor and alteration of Ang II effect in 2K1C rat may be due to upregulation of AT1 receptor.     

Participation of transmembrane proline 82 in angiotensin II AT1 receptor signal transduction

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues. In this project we aimed to investigate the participation of two highly conserved proline residues (Pro82 and Pro162), located in TM II and TM IV, respectively, in AT1 receptor signal transduction. Both mutations did not cause major alterations in AngII affinity. Functional assays indicated that the P162A mutant did not influence the signal transduction. On the other hand, a potent deleterious effect of P82A mutation on signal transduction was observed. We believe that the Pro82 residue is crucial to signal transduction, although it is not possible to say yet if this is due to a direct participation or if due to a structural rearrangement of TM II. In this last hypothesis, the removal of proline residue might be correlated to a removal of a kink, which in turn can be involved in the correct positioning of residues involved in signal transduction.     

Characterization of a specific antibody to the rat angiotensin II AT1 receptor.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)     

Anti-inflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury

Stress reduces gastric blood flow and produces acute gastric mucosal lesions. We studied the role of angiotensin II in gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats were pretreated for 14 days with the AT1 receptor antagonist candesartan before cold-restraint stress. AT1 receptors were localized in the endothelium of arteries in the gastric mucosa and in all gastric layers. AT1 blockade increased gastric blood flow by 40-50%, prevented gastric ulcer formation by 70-80% after cold-restraint stress, reduced the increase in adrenomedullary epinephrine and tyrosine hydroxylase mRNA without preventing the stress-induced increase in adrenal corticosterone, decreased the stress-induced expression of TNF-alpha and that of the adhesion protein ICAM-1 in arterial endothelium, decreased the neutrophil infiltration in the gastric mucosa, and decreased the gastric content of PGE2. AT1 receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, and anti-inflammatory effects (with reduction in TNF-alpha and ICAM-1 expression leading to reduced neutrophil infiltration) while maintaining the protective glucocorticoid effects and PGE2 release. Angiotensin II has a crucial role, through stimulation of AT1 receptors, in the production and progression of stress-induced gastric injury, and AT1 receptor antagonists could be of therapeutic benefit.     

Anti-stress and anti-anxiety effects of centrally acting angiotensin II AT1 receptor antagonists

The brain and the peripheral (hormonal) angiotensin II systems are stimulated during stress. Activation of brain angiotensin II AT(1) receptors is required for the stress-induced hormone secretion, including CRH, ACTH, corticoids and vasopressin, and for stimulation of the central sympathetic activity. Long-term peripheral administration of the angiotensin II AT(1) antagonist candesartan blocks not only peripheral but also brain AT(1) receptors, prevents the hormonal and sympathoadrenal response to isolation stress and prevents the formation of stress-induced gastric ulcers. The mechanisms responsible for the prevention of stress-induced ulcers by the AT(1) receptor antagonist include protection from the stress-induced ischemia and inflammation (neutrophil infiltration and increase in ICAM-1 and TNF-alpha) in the gastric mucosa and a partial blockade of the stress-induced sympathoadrenal stimulation, while the protective effect of the glucocorticoid release during stress is maintained. AT(1) receptor antagonism prevents the stress-induced decrease in cortical CRH(1) and benzodiazepine binding and is anxiolytic. Blockade of brain angiotensin II AT(1) receptors offers a novel therapeutic opportunity for the treatment of anxiety and other stress-related disorders.     

Selective angiotensin II AT2 receptor agonists with reduced CYP 450 inhibition

Structural alterations to the benzylic position of the first drug-like selective angiotensin II AT₂ receptor agonist (1) have been performed, with the emphasis to reduce the CYP 450 inhibitory property of the initial structure. The imidazole moiety, responsible for the CYP 450 inhibitory effect in 1, was replaced with various heterocycles. In addition, the modes of attachment of the heterocycles, that is, carbon versus nitrogen attachment, and introduction of carbonyl functionalities to the benzylic position have been evaluated. In all the three series, AT₂ receptor ligands with affinity in the lower nanomolar range were identified. None of the analogues, regardless of the substituents, exhibited any affinity for the AT₁ receptor. Compounds with substantially reduced inhibition of the CYP 450 enzymes were obtained. Among them the compound 60 was found to induce neurite elongation in NG 108-15 cells and served as potent AT₂ selective agonist.     

The rat angiotensin II AT1A receptor couples with three different signal transduction pathways.

To examine whether the subpopulation of the rat type 1 angiotensin II (AII) receptor (AT1A) couples with a single or multiple signal transduction pathways, we constructed Chinese hamster ovary (CHO) cell lines producing the recombinant receptor. The expressed AT1A receptor exhibits typical pharmacological characteristics of the AT1 receptor, known to mediate the main physiological function of AII. Addition of AII to the CHO cells induced a rapid, transient increase in intracellular free Ca2+ concentrations ([Ca2+]i) followed by a lower, sustained phase. Nicardipine, a blocker of voltage-dependent L-type Ca2+ channels, attenuated the transient [Ca2+]i response and abolished the sustained phase. The transient phase was also reduced dose-dependently by the phospholipase C inhibitor neomycin. Furthermore, AII inhibited forskolin-evoked cAMP accumulation. These data suggest, although another subpopulation named AT1B is present, that the rat AT1A receptor can independently couple with all three signal transduction pathways known to be induced by AII: i.e., i) activation of phospholipase C resulting in InsP3 generation with a subsequent release of intracellularly stored Ca2+, ii) activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels, and iii) inhibition of adenylate cyclase activity.