肾素 - 血管紧张素 - 醛固酮系统阻滞的研究进展

肾素-血管紧张素-醛固酮系统起初被认为是较简单的神经体液调节机制之一。但是,这一想法随着RAAS阻滞剂:肾素阻滞剂、血管紧张素转换酶抑制剂(ACEI)、AT1受体拮抗剂及盐皮质激素受体拮抗剂的深入研究而受到挑战。因此,RAAS的组成、以上药物发挥作用的具体通路及副作用均得到重新定义。在RAAS阻滞剂的应用过程中,机体肾素水平升高,并刺激肾素原受体(即无活性的肾素前体,PRR),进而对机体造成不良影响。同理,在AT1受体拮抗剂的应用过程中,血浆血管紧张素II的水平升高,并与2型血管紧张素II(AT2)受体结合,进而对机体产生有利作用。此外,随着ACEI及ARB的应用,血管紧张素1-7水平升高,其与Mas受体结合,发挥心脏及肾脏保护的作用,还可通过刺激干细胞发挥组织修复作用。     

Concentration-dependent effects of angiotensin II on sinus rate in canine isolated right atrial preparations.

To investigate the concentration-response relationship of angiotensin II with respect to its chronotropic effects, the sinus rate was recorded from canine isolated right atrial preparations perfused through the sinus node artery. Nicotine (5 x 10(-5) M) injection induced an early, atropine-sensitive bradycardic response and a more delayed propranolol-sensitive tachycardic response, suggesting that the preparations contained both cholinergic and adrenergic neurons. The former response, but not the latter, was markedly reduced in preparations in which the right atrial ganglionated plexus was removed. Positive chronotropic responses were induced by angiotensin II over a wide range of concentrations (10(-12) - 5 x 10(-6) M), with a maximum increment of 29.9 +/- 9.6 beats/min. Responses to low concentrations (angiotensin II, 10(-11) M) were monophasic and were abolished by propranolol. In contrast, the responses to higher concentrations (angiotensin II, 10(-6) M) were not abolished by propranolol and were biphasic (early response, 29.9 +/- 12.1 beats/min; later response, 18.6 +/- 9.0 beats/min), the early response being blocked by losartan (AT1 antagonist) but not the later one, both being blocked by saralasin (nonselective angiotensin II antagonist). In conclusion, the data suggest that angiotensin II exerts its stimulant effects on the heart through receptors located either on cardiomyocytes or neurons, depending on the agonist concentration.     

Expression of angiotensin II type 1 receptor in rat hepatic stellate cells and its effects on cell growth and collagen production

OBJECTIVE: To investigate the contribution of angiotensin II towards the process of hepatic fibrosis that is largely due to hepatic stellate cell growth. METHODS: Adult rat hepatic stellate cells were cultured and checked for the expression of angiotensin II receptor 1a (AT(1a)) mRNA by RT-PCR and sequence analysis. The effects of angiotensin II were observed on stimulation of hepatic stellate cell growth detected by MTT assays, (3)H-thymidine incorporation and cell count, and collagen synthesis by (3)H-proline incorporation. RESULTS: We demonstrated that cultured adult rat hepatic stellate cells expressed AT(1a) mRNA, and angiotensin II in a concentration-dependent manner stimulated hepatic stellate cell growth at a concentration of 10(-7)-10(-9) mol/l and collagen synthesis at a concentration of 10(-6)-10(-10) mol/l. Also, AT(1a) receptor antagonist, in a concentration-dependent manner, blocked the cell growth from 10(-6) to 10(-8) mol/l and collagen synthesis from 10(-6) to 10(-9) mol/l. CONCLUSIONS: The results provided direct evidence that AT(1a) mRNA was expressed in rat hepatic stellate cells and angiotensin II could contribute towards the development of hepatic fibrosis via AT(1a) receptor.     

Analysis of the mechanisms underlying the vasorelaxant action of angiotensin II in the isolated rat carotid

It has been suggested that low concentrations of angiotensin II cause vasoconstriction whereas high concentrations evoke vasodilation. Thus, this work aimed to functionally characterize the mechanisms underlying the relaxation induced by angiotensin II at high concentrations in isolated rat carotid rings. Experiments using standard muscle bath procedures showed that angiotensin II (0.01-3 μM) concentration dependently induces relaxation of phenylephrine-pre-contracted rings. No differences between intact or denuded endothelium were found. The angiotensin II-induced relaxation was strongly inhibited by saralasin, the non-selective antagonist of angiotensin II receptors but not by the selective antagonists of AT₁ and AT₂ receptors, losartan and PD123319, respectively. However, A-779, a selective angiotensin-(1-7) receptor antagonist, reduced the relaxation induced by angiotensin II. Administration of exogenous angiotensin-(1-7) on pre-contracted tissues produced concentration-dependent relaxation, which was also inhibited by A-779. HOE-140, the selective antagonist of the bradykinin in B₂ receptor did not produce any significant effect on angiotensin II-induced relaxation. Pre-incubation of denuded-rings with N G-nitro-l-arginine methyl ester (l-NAME) or 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced angiotensin II-induced relaxation. On the other hand, neither indomethacin nor tetraethylammonium (TEA) produced any significant effect. The major new finding of this work is that high concentrations of angiotensin II induce relaxation of the rat carotid via activation of the NO-cGMP pathway through a mechanism that seems to be partially dependent on activation of angiotensin-(1-7) receptors.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

Angiotensin II type 1 (AT1)-receptor blocker prevents impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats

Our aim was to test for smoking-induced endothelial dysfunction in rat cerebral vessels, then to evaluate the effect of valsartan [angiotensin II type I (AT1)-receptor blocker] on that impairment. In pentobarbital-anesthetized, mechanically ventilated Sprague-Dawley rats, we used a cranial window preparation to measure changes in pial vessel diameters following topical applications of acetylcholine (Ach) (before and after smoking or intravenous nicotine infusion; n = 6 in each group), and adenosine (n = 6 for before and after smoking). Then, after intravenous valsartan pretreatment we reexamined the pial vasodilator response to topical Ach (before and after cigarette smoking). Under control conditions, cerebral arterioles were dilated by 6.9 +/- 4.2% and 13.6 +/- 4.8% by topical Ach (10(-6) M and 10(-5) M, respectively) and by 6.4 +/- 2.5% and 12.2 +/- 3.1% by topical adenosine (10(-5) M and 10(-4) M, respectively). One hour after a 1-min inhalation of mainstream smoke (1-mg nicotine cigarette), 10(-5) M Ach constricted cerebral arterioles (-4.4 +/- 4.1%), while 10(-4) M adenosine dilated them by 13.4 +/- 3.4%. One hour after a 1-min nicotine infusion (0.05 mg), 10(-5) M Ach dilated cerebral arterioles by 9.9 +/- 2.4%. Thus, vasodilator response to topical Ach was impaired after smoking, whereas that to adenosine was unaffected. However, the vasodilator response to Ach was unaffected by intravenous nicotine. Valsartan prevented smoking from impairing Ach-induced vasodilation. In conclusion, acute single-cigarette smoking causes a dysfunction of endothelium-dependent, but not endothelium-independent, vasodilation of rat cerebral vessels in vivo, and the effect was not mimicked by intravenous nicotine. AT1-receptor blockade prevented the above smoking-induced impairment of endothelium-dependent vasodilation.     

Further studies on the effect of intracellular angiotensins on heart cell communication: on the role of endogenous angiotensin II

The influence of intracellular angiotensin I (Ang I) and angiotensin II (Ang II) on the process of cell communication was investigated in isolated cell pairs from the failing heart of cardiomyopathic hamsters at 2 and at 6 months of age. Measurements of junctional conductance were performed on weekly coupled ventricular cells (4-5.3 nS) using two separated voltage clamp circuits. The results indicated that at 2 months of age, when no signs of heart failure are detected, the angiotensin converting enzyme (ACE) activity is low and similar to controls (0.26 nmol/mg/min). Here the intracellular dialysis of angiotensin I (10(-8) M) caused a decline of junctional conductance of 33+/-3.6% (n=35) (P<0.05) within 10 min while the administration of the same concentration of Ang I elicited cell uncoupling in cell pairs of 6-month-old cardiomyopathic hamsters in which the ACE activity was enhanced (0.41+/-0.05 nmol/mg/min) (P<0.05). Intracellular administration of angiotensin II in cell pairs of 2-month-old hamsters caused a decline of junctional conductance of only 25+/-4.5% (n=35) (P<0.05) compared to cell uncoupling in 6-month-old cardiomyopathic hamsters. Intracellular losartan(10(-8) M) reduced the effect of intracellular Ang II by 68+/-3.5% (n=28) on 2-month-old hamsters and abolished the effect of the peptide on 6-month-old hamsters. To investigate the influence of endogenous angiotensin II on the regulation of cell coupling, enalapril maleate (10(-8) M) or enalaprilat (10(-9) M) was used. The results indicated that at 2 months of age, no change in cell coupling was elicited by the ACE inhibitor while at 6 months of age, there was an increment of cell coupling of 72+/-6.2% (P<0.05). Similar results were found with intracellular losartan (10(-8) M). These results support the view that endogenous angiotensin II is involved in the regulation of cell communication at an advanced stage of heart failure when the ACE activity is enhanced and the cardiac renin angiotensin system (RAS) is activated.     

ACE inhibition lowers angiotensin II-induced chemokine expression by reduction of NF-kappaB activity and AT1 receptor expression

OBJECTIVE: Angiotensin converting enzyme (ACE) inhibitors significantly improve survival in patients with atherosclerosis. Although ACE inhibitors reduce local angiotensin II (AngII) formation, serine proteases form AngII to an enormous amount independently from ACE. Therefore, our study concentrates on the effect of the ACE-inhibitor ramiprilat on chemokine release, AngII receptor (ATR) expression, and NF-kappaB activity in monocytes stimulated with AngII. METHODS AND RESULTS: AngII-induced upregulation of IL-8 and MCP-1 protein and RNA in monocytes was inhibited by the AT1R-blocker losartan, but not by the AT2R-blocker PD 123.319. Ramiprilat dose-dependently suppressed AngII-induced upregulation of IL-8 and MCP-1. The suppressive effect of ramiprilat on AngII-induced chemokine production and release was in part caused by downregulation of NF-kappaB, but more by a selective and highly significant reduced expression of AT1 receptors as shown in monocytes and endothelial cells. CONCLUSION: In our study we demonstrated for the first time that ramiprilat reduced expression of AT1R in monocytes and endothelial cells. In addition, ramiprilat downregulated NF-kappaB activity and thereby reduced the AngII-induced release of IL-8 and MCP-1 in monocytes. This antiinflammatory effect, at least in part, may contribute to the clinical benefit of the ACE inhibitor in the treatment of coronary artery disease.     

Lisinopril和Losartan对乳鼠心脏成纤维细胞AT1受体基因表达水平和ACE活力的影响

为了解两种降压药物 -血管紧张素转换酶 (angiotensin converting enzyme,ACE)抑制剂(lisinopril)和血管紧张素 型受体 (AT1 R)拮抗剂 (losartan)在心脏间质组织中的作用 ,进行了药物对乳鼠心脏成纤维细胞 AT1 R基因表达和血管紧张素转换酶活力影响的实验 .用 RT- PCR方法检测体外培养乳鼠心脏成纤维细胞 AT1 R基因的表达 .结果显示 ,losartan在 1 0 -7～ 1 0 -4 mol/L浓度范围内对心肌成纤维细胞 AT1 R基因表达有激活作用 ,其中 1 0 -5mol/L浓度激活作用最强 ;1 0 -5mol/L losartan对 AT1 R基因表达呈现明显的时间依赖性变化 ,当加入 1 h后 ,AT1 R基因表达量增加 2倍 ,随后出现一过性下降 ,2 4 h时回升并维持在一较高水平 .与 losartan相比 ,lisinopril对 AT1 R基因的表达无明显影响 .酶活实验结果显示 ,lisinopril明显抑制心肌成纤维细胞中的ACE活力 ,随时间延长 ,酶活力逐渐恢复 (1 2 .6× 1 0 -3 U/mg) ;而 losartan则对酶活力的影响不明显 ,仅是在 1 2 h后 ,ACE活力才略有升高 .实验结果证明 ,在心脏成纤维细胞中存在有 ACE和AT1 R,并且后者受 losartan以浓度和时间依赖方式的调节 .     

Angiotensin II stimulation of the rat pituitary tumoral cell proliferation in vitro.

The effect of angiotensin II (AT II) on proliferation of rat pituitary tumoral cells was investigated in vitro. The tumoral cells were isolated from the prolactin-secreting pituitary tumors induced by stilboestrol implantation. The incorporation of [3H]-thymidine into DNA was used as an index of cell proliferation. It was found that AT II significantly enhanced the [3H]-thymidine incorporation into pituitary tumoral cells in the concentrations of 10(-10) and 10(-8) M. The stimulatory effect disappeared at the concentration of 10(-6) M. The possible involvement of pituitary renin-angiotensin system in pituitary tumorigenesis was discussed.     

Effects of angiotensin II antagonists on the contractile and hydrosmotic effect of AT II and AT III in the toad (Bufo arenarum)

The effects of peptide and non-peptide angiotensin II receptor antagonists on the responses to angiotensin II were examined using aortic rings and skin isolated from the toad. The contractile responses of aortic rings to (Ala-Pro-Gly) angiotensin II were inhibited by the angiotensin II analogue Leu⁸ angiotensin II, with a pA₂ value of 7.6. Similarly, the concentration response curve for (Ala-Pro-Gly) angiotensin II was displaced to the right by the specific angiotensin receptor subtype antagonist DuP 753, with a pA₂ value of 6.0. In contrast, the angiotensin receptor subtype 2 antagonists PD 123177 and CGP 42112A did not modify the contractile response to (Ala-Pro-Gly) angiotensin II. None of the antagonists was able to alter the contractile response to norepinephrine. Both Leu⁸ angiotensin II (10^-8 mol·l^-1) and DuP 753 (10^-6 mol·l^-1) partially inhibited angiotensin III-induced contractions in toad aorta. Angiotensin III, in turn, exhibited lower activity than [Asn¹-Val⁵] angiotensin II in this preparation, its molar potency ratio being 0.293. Previous work from this laboratory reported that osmotic water permeability in the skin of the toad Bufo arenarum was increased by angiotensin II, the effect being blocked by the peptide antagonist Leu⁸ angiotensin II. The hydrosmotic response to [Asn¹-Val⁵] angiotensin II (10^-7 mol·l^-1) was significantly inhibited by DuP 753 (10^-6 and 5×10^-6 mol·l^-1), whereas the response was not inhibited by a tenfold higher concentration of either PD 123177 or CGP 42112A. DuP 753 (10^-6 mol·l^-1) also inhibited the hydrosmotic response to angiotensin III (10^-7 mol·l^-1). These results suggest that receptors for angiotensin II present in isolated toad aorta and skin exhibit pharmacological features similar to those characterized as angiotensin subtype 1 in mammalian tissues.Abbreviations AT ₁ angiotensin receptor subtype 1 - AT ₂ angiotensin receptor subtype 2 - AT II angiotensin II - AT III angiotensin III - CDRC cumulative doseresponse curve(s) - NE norepinephrine - SCC short-circuit current     

Chronic infusion of angiotensin-(1-7) reduces heart angiotensin II levels in rats

We tested the hypothesis that the actions of Angiotensin (Ang)-(1-7) in the heart could involve changes in tissue levels of Ang II. This possibility was addressed by determining the effect of chronic infusion of Ang-(1-7) on plasma and tissue angiotensins. Ang-(1-7) was infused subcutaneously (osmotic minipumps) in Wistar rats. Angiotensins were determined by radioimmunoassay (RIA) in plasma, heart, and kidney. Tissue and plasma angiotensin-converting enzyme (ACE) activity and plasma renin activity (PRA) were also measured. Cardiac and renal ACE2 mRNA levels and cardiac angiotensinogen mRNA levels were assessed by semi-quantitative polymerase chain reaction (PCR). AT1 receptor number was evaluated by autoradiograph. Chronic infusion of Ang-(1-7) (2 microg/h, 6 days) produced a marked decrease of Ang II levels in the heart. A less pronounced but significant decrease of Ang-(1-7) was also observed. No significant changes were observed for Ang I. Ang II was not altered in the kidney. In this tissue, a significant increase of Ang-(1-7) and Ang I concentration was observed. A significant increase of plasma Ang-(1-7) and Ang II was also observed. Ang-(1-7) infusion did not change ACE activity or PRA. A selective slight significant increase in ACE2 expression in the heart was observed. Heart angiotensinogen mRNA as well as the number of Ang II binding sites did not change. These results suggest that AT1 receptors-independent changes in heart Ang II concentration might contribute for the beneficial effects of Ang-(1-7) in the heart. Moreover, these results reinforce the hypothesis that this angiotensin plays an important site-specific role within the renin-angiotensin system.     

Renin-angiotensin system inhibition on noradrenergic nerve terminal function in pacing-induced heart failure

Chronic angiotensin-converting enzyme (ACE) inhibition has been shown to improve cardiac sympathetic nerve terminal function in heart failure. To determine whether similar effects could be produced by angiotensin II AT(1) receptor blockade, we administered the ACE inhibitor quinapril, angiotensin II AT(1) receptor blocker losartan, or both agents together, to rabbits with pacing-induced heart failure. Chronic rapid pacing produced left ventricular dilation and decline of fractional shortening, increased plasma norepinephrine (NE), and caused reductions of myocardial NE uptake activity, NE histofluorescence profile, and tyrosine hydroxylase immunostained profile. Administration of quinapril or losartan retarded the progression of left ventricular dysfunction and attenuated cardiac sympathetic nerve terminal abnormalities in heart failure. Quinapril and losartan together produced greater effects than either agent alone. The effect of renin-angiotensin system inhibition on improvement of left ventricular function and remodeling, however, was not sustained. Our results suggest that the effects of ACE inhibitors are mediated via the reduction of angiotensin II and that angiotensin II plays a pivotal role in modulating cardiac sympathetic nerve terminal function during development of heart failure. The combined effect of ACE inhibition and angiotensin II AT(1) receptor blockade on cardiac sympathetic nerve terminal dysfunction may contribute to the beneficial effects on cardiac function in heart failure.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Angiotensin II receptor subtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skin slices.

Among the many functions of angiotensin II (Ang II) it now appears that Ang II is a growth factor. The concentration of Ang II in rat skin has been shown to increase during wound healing. To investigate the intracellular effect of Ang II in skin we determined the levels of total cytoplasmic inositol phosphates after incubation of skin slices with different doses of Ang II. 10(-6) M of Ang II increased significantly the phosphatidylinositol (PI) hydrolysis, and the effect was dose dependent up to 10(-4) M Ang II. The majority of inositol phosphates yielded after 1 hour incubation in the presence of lithium was InsP1, with lesser amount of InsP2. Losartan, the Ang II AT1 antagonist, at a dose of 10(-4) M blocked the effect of Ang II, while PD123319, the Ang II AT2 antagonist, had no antagonistic action; PD123319 at the higher dose of 10(-3) M, however, potentiated the effect of Ang II on PI hydrolysis. The results suggest that PI hydrolysis is a second messenger system for Ang II in rat skin. Also, the two subtypes of Ang II receptors mediate opposite effects on PI hydrolysis: Ang II binding to AT1 receptors increases inositol phosphate production, while Ang II binding to AT2 receptors decreases inositol phosphate production.     

Molecular mechanisms of angiotensin II stimulation on aquaporin-2 expression and trafficking

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.     

M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M(3) receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M(3)-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M(3)-receptor knockout mice, suggesting that contraction was predominantly due to M(3)-receptor activation. However, approximately 50-60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M(3)-receptor knockout mice, indicating that contraction in these tissues was also due to M(2)-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M(3)-receptor knockout mice by M(1)-receptor activation. Thus M(3)-receptor knockout mice provided unambiguous evidence that M(3) receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M(2) receptors in mouse stomach fundus and trachea.