Comparative distribution of smooth muscle postsynaptic contractile responses in canine trachea and bronchus in vivo

The response of canine tracheal and bronchial smooth muscle to i.a. administered agonists causing smooth muscle contraction was compared in 22 mongrel dogs in vivo. Tracheal and bronchial contractile responses were measured isometrically in situ in the same dogs. In nine dogs, dose-response curves were generated with i.a. acetylcholine and histamine (10(-10) to 10(-6) mol) in a 4-cm tracheal segment and a 1-cm segment of third order bronchus. The tracheal response to i.a. histamine was 36.5 +/- 4.48% of the response to equivalent doses of acetylcholine. In bronchus, the contraction caused by histamine was 81.0 +/- 2.83% of the cholinergic contractile response (P less than .001). In five dogs having beta adrenergic blockade with propranolol, tracheal contraction to 10(-8) to 10(-6) mol i.a. norepinephrine was 27.3 +/- 4.2% of the response to acetylcholine. However, in bronchus, contraction to norepinephrine was 218 +/- 16.6% of the response to equivalent doses of acetylcholine (P less than .001). Phentolamine (200-400 micrograms/kg i.a.) caused 79 to 100% blockade of the tracheal and bronchial response to i.a. norepinephrine. Cholinergic contraction was blocked specifically with 5 micrograms/kg i.a. of atropine. It is concluded that there is substantial heterogeneity in the contractile responses of canine trachea and bronchus in situ. Relative to cholinergic contraction, both histamine and alpha adrenergic stimulation cause substantially greater contraction of airway smooth muscle in the third order bronchus than in trachea.     

Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A

Calyculin-A (CL-A), a novel marine toxin isolated from Discodermia calyx, caused contraction in the smooth muscle of guinea pig taenia ceci and rat aorta in the presence or absence (with 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) of external Ca++ at concentrations ranging from 1 X 10(-8) to 1 X 10(-6) M. In the presence of external Ca++, the contraction induced by CL-A was accompanied by an increase in the cytosolic free Ca++ concentration [( Ca++]cyt) as measured by the fluorescence indicator fura-2. Verapamil (3 X 10(-6) M) inhibited the increase in [Ca++]cyt, but not tension development caused by CL-A. In the absence of external Ca++, CL-A still caused contraction without changing [Ca++]cyt. Thus, from studies with intact smooth muscle it was demonstrated that, in the absence of external Ca++, CL-A can induce a contraction that was not accompanied by an increase in [Ca++]cyt. In permeabilized taenia, CL-A caused contraction in the absence of Ca++ (with 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid) at concentrations similar to those required to contract intact tissue. This contraction was inhibited by the nonselective kinase inhibitors such as amiloride (1 X 10(-3) M) and K-252a (2 X 10(-5) M). Low concentrations of Ca++ (approximately 1 X 10(-6) M) augmented the CL-A-induced contraction in the permeabilized taenia. In native actomyosin prepared from chicken gizzard CL-A induced phosphorylation of the 20 kDa myosin light chain (MLC) in the absence of Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ergometrine on airway smooth muscle contractile responses

A 26-year-old asthmatic female developed severe asthma within a few hours of receiving three oral doses of 0.4 mg ergometrine maleate for the control of postpartum haemorrhaging. This experience and two previous reports of bronchospasm in asthmatic subjects following ergometrine suggested that ergometrine altered airway smooth muscle tone. In the present investigation the effect of ergometrine was studied on canine tracheal smooth muscle strips. Ergometrine (10(-9) M-10(-4) M) induced contraction of canine tracheal smooth muscle. The concentration causing 50% of maximal contraction (EC50) was 4.73 X 10(-8) M. The acetylcholine EC50 was not altered by ergometrine (10(-9) M or 10(-8) M); however, acetylcholine (10(-4) M and 10(-3) M) induced contractions were enhanced by ergometrine (10(-8) M). The data suggest that ergometrine maleate may cause broncho-constriction in some patients with asthma.     

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)     

Pharmacological characterization of the postsynaptic alpha adrenoceptors in vascular smooth muscle from canine and rat mesenteric vascular beds

Postsynaptic alpha adrenoceptors have been characterized in rat superior mesenteric artery and branches of canine mesenteric artery. l-Phenylephrine, naphazoline and clonidine were used as agonists and prazosin and yohimbine were used as antagonists. The pA2 values for prazosin and yohimbine were determined against these agonists in canine and rat mesenteric arteries. The pA2 values for either prazosin or yohimbine were similar when l-phenylephrine and naphazoline were used as the agonists. The slope of the Schild plot was less than unity for prazosin and yohimbine acting as antagonists to l-phenylephrine in canine mesenteric artery whereas in the rat mesenteric artery, the slopes obtained for either phenylephrine or naphazoline against the antagonists were not significantly different from unity. In rat mesenteric artery, the pA2 value for yohimbine against clonidine was not statistically different from values obtained with the other agonists; however, in the presence of prazosin the dose-response curve to clonidine was biphasic, suggesting that the action of clonidine is mediated through two distinct sites to which prazosin has different affinities. However, these results do not provide definitive support for the concept of functionally independent subtypes of postsynaptic alpha adrenoceptors in rat and canine mesenteric artery.     

Mechanism of contractile response to bombesin of the longitudinal muscle from guinea pig ileum

The mechanism of action of bombesin in the longitudinal muscle with myenteric plexus (LM-MP) preparation from guinea pig ileum was examined. Bombesin (3 x 10(-10) to 3 X 10(-7) M) induced contraction in a concentration-dependent manner. The contractions induced by bombesin at concentrations of 10(-9) M and less were tetrodotoxin- and scopolamine-sensitive, and those induced at concentrations of 10(-8) M and greater were tetrodotoxin-sensitive, and partially scopolamine-sensitive. In association with the mechanical response to bombesin, the peptide induced Ca(++)-dependent and tetrodotoxin-sensitive release of acetylcholine from the LM-MP preparation. Bicuculine inhibited partly both the cholinergic contraction and the release of acetylcholine induced by bombesin, suggesting the involvement of a gamma-aminobutyric acid (GABA)-ergic mechanism. Bombesin also induced Ca(++)-dependent and tetrodotoxin-sensitive release of endogenous and [3H]GABA from the LM-MP preparation. These results indicate that bombesin induces contraction mainly mediated mediated by cholinergic and partially mediated by noncholinergic excitatory neurons. GABAergic neurons may be involved in a part of the response to bombesin mediated by the cholinergic neuron.     

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)     

Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster

VSMCs respond to changes in the local environment by adjusting their phenotype from contractile to synthetic, a phenomenon known as phenotypic modulation or switching. Failure of VSMCs to acquire and maintain the contractile phenotype plays a key role in a number of major human diseases, including arteriosclerosis. Although several regulatory circuits that control differentiation of SMCs have been identified, the decisive mechanisms that govern phenotypic modulation remain unknown. Here, we demonstrate that the mouse miR-143/145 cluster, expression of which is confined to SMCs during development, is required for VSMC acquisition of the contractile phenotype. VSMCs from miR-143/145–deficient mice were locked in the synthetic state, which incapacitated their contractile abilities and favored neointimal lesion development. Unbiased high-throughput, quantitative, mass spectrometry–based proteomics using reference mice labeled with stable isotopes allowed identification of miR-143/145 targets; these included angiotensin-converting enzyme (ACE), which might affect both the synthetic phenotype and contractile functions of VSMCs. Pharmacological inhibition of either ACE or the AT₁ receptor partially reversed vascular dysfunction and normalized gene expression in miR-143/145–deficient mice. We conclude that manipulation of miR-143/145 expression may offer a new approach for influencing vascular repair and attenuating arteriosclerotic pathogenesis.     

Relationship between smooth muscle volume and contractile response in airway tissue. Isometric versus isotonic measurement

Two different methods of recording contractile response (isometric and isotonic) were used in both spirally cut and intact ring preparations of rabbit airways. The volume of smooth muscle in each airway preparation was compared with the amount of contraction induced by histamine and carbachol. There were significant relationships between smooth muscle volume and carbachol-induced contraction measured isometrically in spiral and ring preparations. There also was a significant relationship between the proportion of airway smooth muscle and the histamine-induced contraction measured isometrically on ring preparations. There were no significant relationships between smooth muscle volume or proportion of airway smooth muscle and the carbachol or histamine contraction measured isotonically. It is concluded that isometric measurements may provide a more accurate representation of smooth muscle changes in response to agonists in vitro. This may indicate that hyper-responsiveness of airway smooth muscle in vitro is more likely to be detected by isometric rather than isotonic measurements.     

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.     

Relaxant effect of amiloride on canine tracheal smooth muscle

Amiloride, a K+-sparing diuretic, relaxed canine tracheal smooth muscle strips contracted isometrically with high potassium (KCl), carbachol, serotonin and histamine. This indicated that relaxation was not linked to an interaction with an agonist specific receptor. Amiloride-induced relaxation was also not mediated through the production of relaxant prostaglandins, or by the endogenous release of catecholamines. During potassium contractions, amiloride addition produced a slow monophasic, dose-dependent relaxation (IC50 = 12.3 microM). In carbachol contracted strips, 1 and 10 microM amiloride induced a slow monophasic relaxation. With 35 to 250 microM, an initial rapid phase (IC50 = 75.5 microM) was superimposed onto this slow phase (IC50 = 23.5 microM), producing a biphasic relaxation. The rates of relaxation decreased with increased external [Na+] regardless of stimulus, suggesting possible competitive inhibition of a sodium-dependent process. Exposure caused a rapid decline in tension followed by a recovery phase. Tension maintenance during potassium contraction decreased transiently upon the addition of acid to a much lesser extent. Amiloride (100 microM) depressed tension recovery after acid exposure in both cases. Based on the known actions of this drug, inhibition of the Na+-H+ antiporter appears to be consistent with these data. This suggests amiloride may well belong to a new class of smooth muscle relaxants.     

Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells.

Extracellular superoxide was detected in cultures of monkey and human arterial smooth muscle cells as indicated by superoxide dismutase inhibitable reduction of cytochrome c. Superoxide production by these cells in the presence of Fe or Cu resulted in modification of low density lipoprotein (LDL). The degree of LDL modification was directly proportional to the rate of superoxide production by cells. Superoxide dismutase (100 micrograms/ml), and the general free radical scavengers butylated hydroxytoluene and butylated hydroxyanisole (50 microM), inhibited Fe- and Cu-mediated modification of LDL by monkey smooth muscle cells, while catalase (100 micrograms/ml) and mannitol (25 mM) had no effect. The chelators desferrioxamine and diethylenetriamine pentaacetic acid completely inhibited Fe- and Cu-promoted modification of LDL, while EGTA had no inhibitory effect. EDTA stimulated Fe-promoted modification in the 1-100 microM range while inhibiting Cu-mediated modification of LDL. LDL modified by smooth muscle cells in the presence of 10 microM Fe or Cu stimulated [14C]oleate incorporation into cholesteryl ester by human macrophages and murine J774 cells to a degree comparable to that produced by acetylated LDL. LDL incubated with smooth muscle cells and metal ions in the presence of superoxide dismutase failed to enhance macrophage cholesteryl ester accumulation. Thus, arterial smooth muscle cells in culture generate superoxide and modify LDL by a superoxide-dependent, Fe or Cu catalyzed free radical process, resulting in enhanced uptake of the modified LDL by macrophages. Neither hydroxyl radicals nor H2O2 are likely to be involved. Superoxide-dependent lipid peroxidation may contribute to biological modification of LDL, resulting in foam cell formation and atherogenesis.     

Serotonin-induced relaxation in canine coronary artery smooth muscle.

Serotonin (5-HT) is a potent contractile agonist in canine coronary artery devoid of endothelium; however, in higher concentrations 5-HT produces concentration-dependent relaxation by activating an as yet uncharacterized receptor. This study explored the possibility that 5-HT-induced relaxation was mediated by interaction with a member of the 5-HT1, 5-HT2, 5-HT3, or 5-HT4 receptor family. 5-HT, 5-carboxamidotryptamine and 5-methoxytryptamine produced concentration-dependent relaxation in vitro in tissues precontracted with prostaglandin F2 alpha (10 microM). The agonist rank order potency for relaxation was 5-carboxamidotryptamine > 5-HT > 5-MeOT. 8-hydroxydipropylaminotetralin (8-OH-DPAT), dipropyl-5-CT, 5-methyltryptamine, sumatriptan, alpha-methyl-5-HT and 2-methyl-5-HT did not produce significant relaxation. The 5-HT1/beta adrenergic receptor antagonist propranolol (1 microM) did not antagonize 5-HT-induced relaxation. 5-HT-induced relaxation was not blocked by tetrodotoxin (0.3 microM), suggesting that neuronal depolarization to release mediators from nerves was not responsible for the relaxation. Neither ketanserin (1 microM) nor ritanserin (1 microM) antagonized 5-HT-induced relaxation, suggesting that 5-HT2 and 5-HT1C receptors do not mediate relaxation. ICS 205-930 (10 microM), a 5-HT3/5-HT4 receptor antagonist, shifted the 5-HT concentration-response curve modestly to the right (pKB = 5.1 +/- 0.1). Cisapride, a 5-HT4 receptor agonist, was not effective either as an agonist (up to 10 microM), or as an antagonist (1 microM) of 5-HT-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

苦豆子对豚鼠内脏平滑肌收缩活动的干预

目的：观察苦豆子水煎剂对豚鼠离体内脏平滑肌作用的影响，分析其发挥作用途径。 方法：实验于2004—12在宁夏医学院基础学院生理实验室完成。①选用健康成年三色毛豚鼠24只，雌雄不拘。②苦豆子（由银川市中医院提供，由该院检定）被制备成1kg／L的水煎剂，实验的系列质量浓度为1&;#215;10^-4，3&;#215;10^-4，1&;#215;10^-3，3&;#215;10^-3，1&;#215;10^-2，2&;#215;10^-2kg／L。③实验方法：实验时分别取出豚鼠胆囊、胃、小肠及膀胱。每个胆囊制备3条平滑肌肌条，共72条；胃、小肠及膀胱各制备12条平滑肌肌条。累积于置胆囊、胃、小肠及膀胱平滑肌肌条各12条的肌槽中加入1&;#215;10^-4，3&;#215;10^-4，1&;#215;10^-3，3&;#215;10^-3，1&;#215;10^-2，2&;#215;10^-2kg／L苦豆子水煎剂，加药间隔2min。容量50μL。④按随机抽签法将其余60条胆囊肌条分为5组：阿托品＋苦豆子组、酚妥拉明＋苦豆子组、维拉帕米＋苦豆子组、苯海拉明＋苦豆予组、吲哚美辛＋苦豆子组，每组12条。上述5组分别加入拮抗剂阿托品（胆碱能M受体阻断剂）1&;#215;10^-6mol／L，酚妥拉明（肾上腺素能α受体阻断剂）1&;#215;10^6mol／L，维拉帕米（Ca^2＋拮抗剂）1&;#215;10^-7mol／L，苯海拉明（组织胺H1受体阻断剂）1&;#215;10^-6mol／L，吲哚美辛（前列腺隶受体阻断剂）1&;#215;10^6mol／L后2min再依次加入前述“累积加药”中各质量浓度苦豆子。⑤通过JH-2肌肉张力换能器将信号输出BL-410生物机能实验系统读出标本的张力、收缩波平均振幅及收缩频率的变化值。⑥数据间差异性比较采用均数t检验。 结果：①累积加入苦豆子水煎剂1&;#215;10^-4，3&;#215;10^-4，1&;#215;10^-3，3&;#215;10^-3，1&;#215;10^-2.2&;#215;10^-2kg／L后，胆囊肌条张力逐渐增高、收缩频率逐渐加快、收缩波平均振幅逐渐减小。（函当苦豆子水煎剂累积质量浓度为2&;#215;10^-2kg／L时，酚妥拉明＋苦豆子组离体胆囊肌条张力和收缩频率明显低于或慢于苦豆子组（t=2，3401，2．1404，P〈0．05），而收缩波平均振幅明显高于苦豆子组（t=2．8256，P〈0．05）。维拉帕米＋苦夏子组离体胆囊肌条张力明显低于苦豆子组（t=2．3464，P〈0．05）。③当苦豆子水煎剂累积质量浓度为1&;#215;10^-2kg／L时，酚妥拉明＋苦夏子组离体胆囊肌条张力明显低于苦豆子组（t=2．430，P〈0．05），而收缩波平均振幅明显高于苦豆子组（t=2．2279，P〈0．05）。维拉帕米＋苦豆子组离体胆囊肌条张力明显低于苦豆子组（t=2．6576，P〈0．05）。④累积加入不同质量浓度苦赢子水煎剂后对豚鼠胃、小肠及膀胱肌条的张力、收缩波平均振幅及收缩频率无明显影响（P〉0．05）。 结论：①苦豆子水煎刺可呈剂量依赖性增高胆囊平滑肌肌条的张力、加快收缩频率、减小收缩波平均振幅，其作用可被酚妥拉明及维拉帕米部分阻断，且该种作用可能与胆囊平滑肌细胞膜上的肾上腺素能Ⅱ受体和细胞膜上的Ca^2＋通道有关。②苦豆子水煎剂对豚鼠胃、小肠及膀胱平滑肌的收缩活动无明显影响。     

Insulin attenuates vasopressin-induced calcium transients and a voltage-dependent calcium response in rat vascular smooth muscle cells.

Insulin attenuates the contractile responses of vascular smooth muscle (VSM) to various agonists. Insulinopenic and insulin-resistant rats lack this normal attenuation of vascular contractile responses. To study this attenuating mechanism, the effects of insulin on calcium (Ca2+) responses of cultured VSM cells (a7r5) to arginine vasopressin (AVP) and membrane potential were investigated. Insulin (1 and 100 mU/ml) shifted AVP dose-response curves to the right, reducing relative potency of AVP by 16-fold and 220-fold, respectively. Responses to AVP were significantly attenuated within 30 min of insulin application. The AVP-elicited rise in [Ca2+]i was partially dependent upon extracellular Ca2+. AVP-elicited inward current was reduced by 90 min of insulin treatment (100 mU/ml), from a peak current of -103 +/- 27 pA (normal) to -37 +/- 15 pA (insulin treated). Peak voltage-dependent Ca(2+)-dependent inward current was unaffected by insulin; however, the current-voltage curve was shifted 16 +/-3 mV to the right by insulin. Thus, insulin may reduce VSM contractile responses by attenuating agonist-mediated rises in [Ca2+]i mediated, in part, by reductions in Ca2+ influx through both receptor- and voltage-operated channels.