Comparison of the serotonin receptors that mediate smooth muscle contraction in canine and porcine coronary artery.

Serotonin (5-HT) is one important mediator of the coronary vasospasm and occlusion associated with thrombosis and atherosclerosis. 5-HT concentration-dependently contracted both canine and porcine coronary artery rings in vitro. In the dog, 5-HT-induced contraction was not blocked by either LY53857 (1 microM) or ketanserin (1 microM), but was blocked by the nonselective 5-HT receptor antagonist 1-naphthylpiperazine (1-NP) (100 nM), indicating 5-HT receptor involvement. Unlike the dog, both LY53857 (1 microM) and ketanserin (30 nM) antagonized 5-HT-induced contraction in pig arteries. Dissociation constants for LY53857 and ketanserin in porcine arteries were compared with those in rat jugular vein, a tissue possessing a well characterized 5-HT2 receptor. Both LY53857 (3 nM) and ketanserin (3 nM) antagonized 5-HT-induced contraction in rat jugular vein; however, the affinities of LY53857 and ketanserin in the rat jugular vein were significantly higher than those in the pig coronary. The rank order contractile potency for 5-HT, (alpha Me-5-HT) and sumatriptan in porcine coronary artery was consistent with that established for a 5-HT2 receptor, whereas the rank order potency in canine coronary artery indicated non-5-HT2 receptor involvement. Sumatriptan, a 5-HT1D receptor-selective agonist, was equieffective to 5-HT in contracting the canine coronary artery, a response inhibited by 1-NP (100 nM). Sumatriptan failed to contract either the pig coronary or rat jugular vein. In summary, significant differences exist in the 5-HT receptors that mediate contraction between the canine and porcine coronary artery.(ABSTRACT TRUNCATED AT 250 WORDS)     

M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle.

Classification of muscarinic receptors in the central airways has revealed the coexistence of M2 and M3 muscarinic receptors in this tissue, with the M2 subtype being predominant. Although M3 muscarinic receptors have been linked to airway smooth muscle contraction, a functional role for the M2 subtype in this tissue has been unclear. In nonairway smooth muscle, stimulation of the M2 muscarinic receptor has been shown to be associated with inhibition of adenylyl cyclase. In the present study, characterization of muscarinic receptors in canine tracheal smooth muscle confirmed that the majority of these muscarinic receptors were of the M2 subtype (89 +/- 3%), with a minor population of M3 receptors (11 +/- 3%). In functional studies, both isoproterenol and forskolin cause a dose-dependent relaxation of precontracted airway smooth muscle. In tissues precontracted with methacholine, 11-([[2-(diethylamino)methyl]-1-piperidinyl]acetyl)5,11- dihydro-6H-pyrido[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116), a selective M2 antagonist, shifted dose-response curves to both isoproterenol and forskolin significantly to the left. In contrast, AF-DX 116 did not alter relaxation induced by the K+ channel opener BRL 38227. Furthermore, the ability of AF-DX 116 to enhance isoproterenol-induced relaxation appears to be limited to smooth muscle precontracted with muscarinic agonists because AF-DX 116 had no effect on isoproterenol dose-response curves in muscle strips precontracted with histamine. Hexahydrosiladifenidol (HHSiD), a selective antagonist for M3 receptors, did not shift the isoproterenol dose-response curve in muscle precontracted with methacholine. This study demonstrates that stimulation of M2 muscarinic receptors in canine airway smooth muscle plays an important role in functional antagonism by reducing the relaxation caused by agents such as isoproterenol and forskolin.     

Comparison of serotonergic receptor subtypes on the smooth muscle and endothelium of the canine coronary artery

To characterize the responses of the canine coronary artery to serotonin, rings with and without endothelium were suspended in organ chambers for isometric tension recording. Serotonin evoked an endothelium-dependent relaxation of prostaglandin F2 alpha-contracted rings which was inhibited by antagonists with affinity for 5-hydroxytryptamine (5-HT)1 and 5-HT2 receptors, methiothepin and metergoline, but was not mimicked or antagonized by the 5-HT1A-selective ligand, 8-hydroxy-2-di-n-propylamino tetralin. This relaxation is not mediated by 5-HT1B receptors as it was not antagonized by cyanopindolol; similarly, lack of inhibition by ketanserin and MDL 72222 rule out contributions of 5-HT2 receptors or 5-HT3 receptors. Rings without endothelium contracted to serotonin; this contraction was not blocked by cyanopindolol and was only weakly inhibited by ketanserin, but was antagonized in an apparently competitive fashion by methiothepin and was mimicked by 8-hydroxy-2-di-n-propylamino tetralin (although at higher concentrations than would be expected for its action at a 5-HT1A receptor). At high concentrations, serotonin evoked a relaxation of endothelium-denuded rings, which was blocked by very low concentrations of methiothepin but was unaffected by ketanserin or cyanopindolol. Thus, there appear to be three different serotonergic receptors in the coronary artery. Although available agents do not allow their precise classification as yet, none of them is of the 5-HT2 type.     

Neurogenic cholinergic prejunctional inhibition of sympathetic beta adrenergic relaxation in the canine coronary artery

Electrical stimulation of isolated canine coronary arteries causes release of norepinephrine and subsequent relaxation by activation of beta adrenoceptors. The purpose of the present study was to determine if this beta adrenergic relaxation was influenced by a concomitant release of acetylcholine. Rings of epicardial coronary arteries of the dog were studied in organ chambers filled with physiological salt solution. The tetrodotoxin-sensitive, beta adrenergically mediated relaxation induced by electrical stimulation was studied during contractions evoked by prostaglandin F2 alpha. The relaxation to low-frequency stimulation was inhibited and augmented, respectively, by acetylcholine and atropine, suggesting that release of acetylcholine may modulate the beta adrenergic response to sympathetic nerve stimulation. The relaxation caused by high-frequency stimulation was not affected by atropine or removal of the endothelium, indicating that endogenously released acetylcholine does not act directly on the smooth muscle or initiate an endothelium-dependent vasodilator response. In superfused strips of coronary artery preincubated in [3H]norepinephrine, acetylcholine depressed the stimulated overflow of [3H]norepinephrine, indicating prejunctional cholinergic receptors on adrenergic nerve endings. Atropine augmented the overflow, suggesting that endogenous acetylcholine, released during stimulation, inhibits the release of norepinephrine. These observations suggest that prejunctional inhibition of norepinephrine release, which limits the sympathetic beta adrenergic relaxation of the smooth muscle, is the primary neurogenic cholinergic effect in canine epicardial coronary arteries.     

连接蛋白 Cx43参与冠状动脉平滑肌细胞的异质性促进冠脉再狭窄

目的：研究猪冠状动脉平滑肌细胞异质性与连接蛋白 Cx43关系,进一步阐明连接蛋白 Cx43在冠状动脉再狭窄发生中的作用。方法分别培养正常及支架术后猪原代冠状动脉平滑肌细胞,正常组应用血小板源生长因子（PDGF-BB）进行诱导,两组细胞分别进行电子显微镜观察细胞形态及结构变化,采用 Western Blotting 方法及实时荧光定量 PCR 测定 Cx43、Cx40、α-SMA、S100A4蛋白和 mRNA 表达;然后诱导组应用 Cx43阻断剂进行干预,再次检测上述指标变化。结果正常冠状动脉平滑肌细胞可见：纺锤型（spindle-shaped,S-SMC）和长菱形（rhomboid,R-SMC）两类,以 S-SMC 为主,其内 Cx40、α-SMA 有较高的蛋白和 mRNA 表达,而 Cx43表达较少;正常组经 PDGF-BB 诱导后细胞由 S-SMC 向 R-SMC 转变,同时伴随 Cx43表达上调,而 Cx40表达明显下降;通过反义 RNA 降低 Cx43表达,这种变化受抑制,并且 S-SMC 细胞保持原有形态,同时表达 a-肌动蛋白,支架组平滑肌细胞以 R-SMC 为主,Cx43、S100A4有较高的蛋白和 mRNA 表达。结论冠状动脉平滑肌细胞表型变化与连接蛋白 Cx43表达密切相关,连接蛋白 Cx43可能参与冠脉再狭窄的过程。     

Evidence against serotonin-induced endothelium-dependent relaxation in bovine coronary artery.

Bovine coronary artery rings were mounted in tissue baths for the measurement of isometric contraction in order to test for serotonin-induced endothelium-dependent relaxation. A23187 caused a concentration-dependent relaxation in precontracted artery rings when the endothelium was intact, but not when it was removed. Endothelium removal had no effect on serotonin concentration contractile response curves (CRC) in normal Krebs' solution, but enhanced the response to serotonin in artery rings precontracted with 25 mM K+ Krebs' solution. In endothelium-intact, precontracted artery rings, ketanserin shifted the serotonin CRC to the right, but did not convert the contraction to relaxation. 5-Carboxamidotryptamine caused a weak contraction that was blocked by ketanserin, but not converted to relaxation. Inactivation of monoamine oxidase enhanced the contractile response to serotonin in precontracted artery rings in a manner identical to that caused by endothelium removal. Inactivation of monoamine oxidase had no effect in endothelium-denuded artery rings. De-endothelialized rabbit aorta strips pretreated with phenoxybenzamine were placed in close apposition to endothelium-intact coronary artery strips. Superoxide dismutase, acetylcholine and A23187 caused the precontracted rabbit aorta strip to relax, whereas serotonin had no effect. These results support the conclusion that the endothelium of the bovine coronary artery releases a relaxing factor both spontaneously and in response to acetylcholine and A23187. Serotonin appears to be incapable of releasing endothelium-derived relaxing factor or causing an endothelium-dependent relaxation. However, bovine coronary artery endothelium appears to be able to take up and metabolize serotonin.     

Subcellular localization of tissue factor and human coronary artery smooth muscle cell migration

Summary. Background: Tissue factor (TF) is the most relevant physiological trigger of thrombosis. Additionally TF is a transmembrane receptor with cell signaling functions. Objectives: The aim of this study was to investigate TF subcellular localization, function and signaling in human coronary artery smooth muscle cell migration. Methods: Coronary arteries and primary cultures of vascular smooth muscle cells (HVSMC) were obtained from human explanted hearts. Wound repair and Boyden chamber assays were used to measure migration in vitro. TF‐pro‐coagulant activity (TF‐PCA) was measured in extracted cellular membranes. Analysis of TF distribution was performed by confocal microscopy. A nucleofector device was used for TF and protease activated receptor 2 (PAR2) silencing. mRNA levels were analyzed by RT‐PCR. Results: In migrating HVSMC TF translocates to the leading edge of the cells showing an intense patch‐like staining in the lamellipodia. In the migrating front TF colocalizes with filamin (FLN) in the polarized lipid rafts. TF‐PCA was increased in migrating cells. Silencing of the TF gene inhibits RSK‐induced FLN‐Ser‐2152 phosphorylation, down‐regulates CDC42, RhoA, and Rac1 protein expression and significantly inhibits cell migration. Silencing PAR2 also inhibits cell migration; however, silencing both TF and PAR2 induces a significantly higher effect on migration. Smooth muscle cells expressing TF have been identified in non‐lipid‐rich human coronary artery atherosclerotic plaques. Conclusions: TF translocates to the cell front in association with cytoskeleton proteins and regulates HVSMC migration by mechanisms dependent and independent of factor (F)VIIa/PAR2. These results extend the functional role of TF to smooth muscle cell trafficking in vessel wall remodeling.     

Serotonin-induced Na+/K+ pump stimulation in vascular smooth muscle cells. Evidence for coupling to multiple receptor mechanisms.

Exposure of cultured canine femoral artery vascular smooth muscle cells to serotonin (5-HT) caused a 3.6-fold stimulation of ouabain-sensitive 86Rb uptake. The 5-HT2 receptor antagonist, ketanserin, partly blocked the 5-HT-mediated Na+/K+ pump stimulation and the 5-HT1/5-HT2 receptor antagonist, methiothepin, completely blocked the response, suggesting that both 5-HT1 and 5-HT2 receptors play a role in the 5-HT-mediated Na+/K+ pump activation. Second messengers generated by 5-HT2 receptor-mediated phosphoinositide hydrolysis, Ins(1,4,5)P3 and diacylglycerol were implicated in the stimulatory action of 5-HT on the vascular Na+/K+ pump. Like some other contractile agonists, 5-HT activated a Na+ influx pathway which caused Na+/K+ pump stimulation by increasing the rate-limiting substrate. The maximum stimulation of Na+ influx by 5-HT was 2.5-fold. The 5-HT-stimulated Na+ influx was totally blocked by methiothepin but only 29% inhibited by ketanserin, indicating that most of the Na+ influx was mediated by the 5-HT1 receptor. The 5-HT-stimulated Na+ influx was substantially inhibited by 50 microM dimethylamiloride, suggesting that the Na+ influx pathway stimulated by 5-HT was Na+/H+ exchange. BAPTA/AM 1,2-[bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl) ester], an intracellular Ca++ chelator, partly blocked 5-HT-stimulated Na+ influx and ouabain-sensitive 86Rb uptake, suggesting that Ca++ is an important mediator of these responses. These data suggest that: 1) 5-HT, in addition to its well known activity as a contractile agonist, can stimulate the electrogenic Na+/K+ pump which, in theory, would tend to oppose contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of neuromedin U effects in canine smooth muscle

Two endogenous receptors for the potent smooth muscle-stimulating peptide neuromedin U (NmU) have recently been identified and cloned. Pharmacological, binding, and expression studies were conducted in an attempt to determine the receptor(s) involved in the smooth muscle-stimulating effects of NmU. The NmU peptides caused a concentration-dependent contraction of canine isolated urinary bladder. NmU did not have this same effect in the urinary bladder from rat, guinea pig, rabbit, mouse, or ferret. Although NmU had no effect on canine uterus it did cause contraction of canine stomach, ileum, and colon. As well as causing contraction of canine bladder in vitro, NmU administered systemically resulted in a significant increase in urinary bladder pressure in vivo. High-affinity binding sites for NmU were identified in canine bladder. The four NmU peptides porcine NmU-8, rat NmU-23, human NmU-25, and porcine NmU-25 displaced (125)I-NmU-25 binding with similar K(i) values (0.08-0.24 nM). A different binding profile was revealed in human embryonic kidney-293 cells transiently expressed with the canine NmU-2 receptor where porcine NmU-8 (K(i) = 147.06 nM) was much less potent than the other NmU peptides. Using TaqMan, expression of NmU-1 was detected in human urinary bladder, small intestine, colon, and uterus. Expression of NmU-2 was much lower or absent in these human tissues and undetectable in canine bladder and stomach. The results of this study reveal significant species differences in the activity of NmU. The contractile activity in human and canine smooth muscle seems to be mediated by the recently cloned NmU-1 receptor.     

Potassium channel openers and vascular smooth muscle relaxation

Potassium channel openers comprise a diverse group of chemical agents which open plasma-lemmal K-channels. They show selectivity for smooth muscle, although K-channels in cardiac and skeletal muscle, neurones and the pancreatic beta-cell are also affected at relatively high concentrations. In addition, at least one endogenous K-channel opener of vascular origin--endothelium-derived hyperpolarizing factor--exists and in man plays a role in modulating blood vessel tone. The type of K-channel involved in the actions of both exogenous and endogenous K-channel openers is still uncertain, although a prime candidate in smooth muscle seems similar to the [ATPi]-modulated K-channel in the pancreatic beta-cell. This review focuses attention on the action of these agents in vascular smooth muscle and on the possible clinical exploitation of their powerful vasorelaxant properties.     

Regional differences in relaxation of electric field-stimulated canine airway smooth muscle by verapamil, isoproterenol and prostaglandin E2

Regional differences in contraction produced by methacholine and electric field stimulation (EFS) and in relaxation produced by isoproterenol, prostaglandin E2 and verapamil were studied in isolated canine airway smooth muscle in vitro. Low-frequency EFS (3 Hz, 0.5 msec, 50 V) contracted thoracic trachealis to 43% of maximal EFS response, whereas cervical trachealis contracted to only 14% of maximum. EFS at 10 Hz produced 75% of the maximal response in both regions of the trachea. These EFS responses were abolished by 0.1 microM tetrodotoxin and 1.0 microM atropine. Contraction produced by EFS was also matched in each tissue by contraction with methacholine. The concentrations of methacholine that matched EFS at 10 Hz were 52 +/- 7, 378 +/- 84 and 66 +/- 11 nM for cervical and thoracic trachealis and lobar bronchi, respectively. Both EFS and matched methacholine contractions of cervical trachealis and lobar bronchi were completely relaxed by isoproterenol, whereas thoracic trachealis relaxed maximally to only 60% of induced tone. When verapamil was used to relax EFS and matched methacholine contractions, cervical trachealis was completely relaxed whereas thoracic trachealis relaxed to 15% of induced tone. Although there was a regional difference in the relaxant potency of isoproterenol and, to some extent, verapamil, there was no difference in isoproterenol or verapamil EC50 values for EFS vs. matched methacholine contractions within each region. In contrast, EFS contractions of thoracic trachealis were more sensitive to prostaglandin E2-induced relaxation than were matched methacholine contractions. These data demonstrate marked differences in cholinergic and beta adrenergic receptor-mediated responses between regions of the tracheobronchial tree.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen receptor alpha mediates acute potassium channel stimulation in human coronary artery smooth muscle cells

The pleiotropic effects of estrogen are mediated via stimulation of two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Although a number of studies have identified expression of one or both subtypes in estrogen target tissues, fewer studies have correlated ER expression with a functional role of these proteins in regulating cellular excitability. In the present study, we have combined cellular fluorescence, immunocytochemistry, and molecular expression techniques with single-channel patch-clamp studies to determine which ER mediates estrogen-stimulated potassium channel activity in human coronary artery smooth muscle cells (HCASMC). We had demonstrated previously that estrogen stimulates activity of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel in HCASMC via a nongenomic mechanism. We now demonstrate expression of both ERalpha and ERbeta subtypes in HCASMC. Functionally, however, expression of ERalpha antisense plasmid abolished the acute effect of estrogen on these channels, whereas estrogen retained its ability to stimulate BK(Ca) channels in cells transfected with only green fluorescence protein. In contrast, overexpression of ERalpha enhanced the stimulatory action of estrogen in HCASMC. Transfection with ERalpha antisense/sense plasmid did not alter ERbeta expression. These findings indicate that the ERalpha isoform mediates estrogen-induced stimulation of BK(Ca) channels in HCASMC and thereby provide evidence for a receptor-dependent signaling mechanism that can mediate estrogen-induced inhibition of cellular excitability.     

Adrenoceptor-mediated mechanical responses of canine tracheal smooth muscle

The effect of tone on responses of canine tracheal smooth muscle (TSM) to norepinephrine (NE) was studied to elucidate the role of sympathetic innervation and adrenoceptors in the control of the airways. Electrical field stimulation produced contraction of TSM in vitro which was augmented by eserine, depressed by phentolamine, potentiated by propranolol in the presence of K+ (14 mM) and almost eliminated by tetrodotoxin or atropine. Resting TSM did not contract in response to NE (10(-8) to 10(-4) M) in the presence or absence of propranolol (10(-5)M). The addition of NE (10(-8) to 10(-6) M) at the plateau of contraction produced by K+ (22.8 mM), histamine (10(-6) M) or acetylcholine (5 X 10(-8) M) produced a further phentolamine-sensitive contraction which was potentiated by beta adrenoceptor blockade with propranolol (10(-5) M). The addition of tyramine (10(-5) to 10(-4) M) at the plateau of contraction produced by K+ (22.8 mM) produced a further contraction which was potentiated by propranolol (10(-5) M) and reduced by phentolamine (10(-5) M). Although the response to NE in the presence of elevated tone was contractile at low concentrations of NE (10(-8) to 10(-6) M), a propranolol-sensitive relaxant response was elicited at higher NE concentrations (10(-5) and 10(-4) M). Maximum contractions to NE in the absence or presence of beta-blockade were dependent on the tone of the muscle. These findings suggest a functional adrenergic innervation of canine TSM and the presence of alpha and beta adrenoceptors which mediate contractile and relaxant responses, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of adenylate cyclase attenuates adenosine receptor-mediated relaxation in coronary artery.

This study was designed to evaluate whether the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) would attenuate the relaxation produced by adenosine analogs in order to provide functional evidence in support of the working hypothesis that adenosine receptor-mediated relaxation of coronary artery involves adenylate cyclase. Rings from porcine left anterior descending coronary artery were mounted in organ chambers for measurement of isometric force. Rings contracted with KCl (30 mM) relaxed in a concentration-dependent manner to 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenosine (NECA), isoproterenol, sodium nitroprusside (SNP) and forskolin. Treatment of coronary rings with DDA (50 microM) significantly attenuated the relaxation produced by CAD, NECA, forskolin and isoproterenol, but had no effect on the relaxation response to SNP. The nucleoside transport inhibitor dilazep (10 microM) completely reversed the inhibitory effect of DDA on the relaxation produced by forskolin and CAD, whereas dilazep only partially reversed the DDA inhibition of NECA-induced relaxation. In a membrane preparation from porcine coronary artery CAD, but not NECA, increased cyclic AMP production in a GTP-dependent manner. DDA significantly decreased basal cyclic AMP production and also decreased CAD-, forskolin-, GTP- and NaF-stimulated cyclic AMP production. These results provide functional and biochemical evidence in support of the working hypothesis that adenosine receptor-mediated coronary relaxation involves adenylate cyclase. Furthermore, the results from this study suggest that the signaling mechanisms responsible for adenosine receptor-mediated coronary relaxation are more complicated than a single receptor coupled with adenylate cyclase because 1) dilazep completely reversed the inhibitory effect of DDA on the CAD relaxation but not the NECA relaxation, and 2) NECA did not increase cyclic AMP production.     

Relationship between cyclic nucleotide levels and drug-induced relaxation of smooth muscle.

The anticonvulsant effects of four benzhydryl piperazines, SC-13504 (ropizine, an anticonvulsant), hydroxyzine (HDX, an anxiolytic), chlorcyclizine (CCZ, an antihistaminic) and buclizine (BUC, an antihistaminic), were investigated utilizing a modified maximal electroshock seizure test in rats. In addition to detecting the presence or absence of tonic hindlimb extension, the modified method quantified various phases of the seizure. All four benzhydryl piperazines exhibited anticonvulsant activity in maximal electroshock seizure, but SC-13504 was similar in efficacy to phenobarbital and phenytoin, and much more effective than HDX, CCZ or BUC. Additionally, SC-13504 possessed a therapeutic index much greater than any of the compounds tested. The duration of action of the benzhydryl piperazines, in hours was: SC-13504, 0.5 to 8; HDX, 0.5 to 2; CCZ, 0.5 to 16; and BUC, 2 to 8. Buc and CCZ are postulated to be converted to active anticonvulsant metabolites.     

Age-related decrease in beta adrenergic receptor-mediated vascular smooth muscle relaxation

Beta adrenergic receptor-mediated vascular smooth muscle relaxation decreases with increasing age. We have examined the mechanism responsible for this phenomenon using rat mesenteric arteries from young (5-6 weeks) and older (10-12 months) rats. The beta adrenergic agonist isoproterenol produced a dose-dependent relaxation of serotonin-constricted mesenteric artery rings from young rats, whereas the maximal ability of isoproterenol to relax arterial rings from the older rats was found to be reduced markedly (92.7 vs. 27.6%, P less than .0001). The relaxation responses caused by acetylcholine and nitroglycerin, which appear to act independently of cyclic AMP (cAMP), are similar in the two groups. The loss in responsiveness of the mesenteric artery to isoproterenol was not explained by a change in beta receptor number in the vessels (29 +/- 4 in young rats vs. 31 +/- 7 fmol/mg of protein in the older rats). The maximal stimulation of cAMP accumulation by isoproterenol was lower in the older vessels; forskolin activated cAMP accumulation equally in the two groups. However, the vessels from the older rats were less sensitive to forskolin-induced vascular relaxation. Also, the ability of dibutyryl cAMP to promote vascular relaxation was diminished in the older vessels. These data suggest that the diminished cAMP accumulation in older vessels in response to isoproterenol might not necessarily in itself explain completely the reduced physiological response and that an additional defect in the beta adrenergic-mediated relaxation in the vascular smooth muscle of older rats may lie at the level of cAMP-dependent protein kinase activation or more distally.