MaxiK channel mediates beta2-adrenoceptor-activated relaxation to isoprenaline through cAMP-dependent and -independent mechanisms in guinea-pig tracheal smooth muscle.

We examined the contribution of large-conductance, Ca(2+)-sensitive K+ (MaxiK) channel to beta2-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between beta2-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through beta2-type of adrenoceptor since the response was antagonized in a competitive fashion by a beta2-selective adrenoceptor antagonist butoxamine with a pA2 value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 microM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 microM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 microM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX, 5 microg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein Gs. These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates beta2-adrenoceptor-activated relaxation; 2) both cAMP-dependent and -independent mechanisms underlie the functional coupling between beta2-adrenoceptor and MaxiK channel to induce muscle relaxation; and 3) direct regulation of MaxiK channel by Gs operates in cAMP-independent coupling between beta2-adrenoceptor and this ion channel.     

Functional contribution of voltage-dependent and Ca2+ activated K+ (BK(Ca)) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides.

We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP > CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.     

Studies on the mechanisms underlying beta-adrenoceptor-mediated relaxation of rat abdominal aorta.

Mechanisms underlying beta-adrenoceptor (beta-AR)-mediated vascular relaxation were studied in the isolated rat abdominal aorta. In the endothelium-denuded helical preparations, a non-selective beta-AR agonist isoprenaline elicited a concentration-dependent relaxation. In the absence of beta-AR antagonists, isoprenaline-induced relaxation was not practically affected by an adenylyl cyclase inhibitor SQ 22,536 (300 microM), but was strongly diminished by high-KCl (80 mM). Isoprenaline-induced relaxation in the presence of SQ 22,536 was significantly diminished by iberiotoxin (IbTx, 0.1 microM), but was not affected by 4-aminopyridine (4-AP, 3 mM). Isoprenaline-induced relaxation was not also affected by SQ 22,536 (300 microM) even in the presence of CGP20712A (a beta(1)-selective antagonist) and ICI-118,551 (a beta(2)-selective antagonist) (0.1 microM for each), but was strongly diminished by high-KCl. By contrast, SQ 22,536-resistant, isoprenaline-induced relaxation in the presence of CGP20712A plus ICI-118,551 was not affected by IbTx (0.1 microM), but was inhibited significantly by 4-AP (3 mM). These results suggest that in rat abdominal aortic smooth muscle: 1) both beta(1)-/beta(2)-AR- and beta(3)-AR-mediated relaxations substantially involve cAMP-independent mechanisms; 2) beta(1)-/beta(2)-AR-mediated, cAMP-independent relaxant mechanisms are partly attributed to the large-conductance, Ca (2+)-sensitive K(+) (MaxiK, BK) channel whereas beta(3)-AR-mediated relaxant mechanisms are attributed to K(v) channel.     

MaxiK channel-triggered negative feedback system is preserved in the urinary bladder smooth muscle from streptozotocin-induced diabetic rats.

MaxiK channel, the large-conductance Ca2+-sensitive K+ channel, facilitates a negative feedback mechanism to oppose excitation and contraction in various types of smooth muscles including urinary bladder smooth muscle (UBSM). In this study, we investigated how the contribution of MaxiK channel to the regulation of basal UBSM mechanical activity is altered in streptozotocin-induced diabetic rats. Although the urinary bladder preparations from both control and diabetic rats were almost quiescent in their basal mechanical activities, they generated spontaneous rhythmic contractions in response to a MaxiK channel blocker, iberiotoxin (IbTx). The effect of IbTx on the mechanical activity was significantly greater in diabetic rat than in control animal. Similarly, the basal mechanical activity was increased with apamin, an inhibitor for some types of small conductance Ca2+-sensitive K+ channels, and this effect was more pronounced for diabetic rat. However, in both control and diabetic animals, IbTx action was stronger than that of apamin. Diabetes also enhanced the responses to BayK 8644, an L-type Ca2+ channel agonist. The extent of this enhancement in diabetic bladder vs. control was, however, almost the same as that attained with IbTx. Expression levels for MaxiK channel as well as apamin-sensitive K+ channels and L-type Ca2+ channel were not altered by diabetes, when determined as their corresponding mRNA levels. These results indicate that diabetes can potentially increase the basal UBSM mechanical activity. However, in diabetic UBSM, the main negative-feedback system triggered by MaxiK channel is still preserved enough to counteract the possible enhancement of this smooth muscle mechanical activity.     

Adrenaline produces the relaxation of guinea-pig airway smooth muscle primarily through the mediation of beta(2)-adrenoceptors.

The beta-adrenoceptor subtype that mediates adrenaline-induced relaxation was pharmacologically identified in smooth muscle cells of the isolated guinea-pig trachea. Adrenaline produced a concentration-dependent relaxation with a pD(2) value of 7.1. The concentration-response curve for adrenaline was shifted rightwards in a competitive fashion by the beta(1)-/beta(2)-nonselective antagonists propranolol and bupranolol, with pA(2) values of 8.85 and 8.97, respectively. Adrenaline-induced relaxation was not affected by the beta(1)-selective antagonists atenolol and CGP-20, 712A within the concentration ranges supposed to antagonize the beta(1)-subtype (atenolol, or=3x10(-6) M with a pA(2) value of 5.77. The concentration-response curve for adrenaline was also competitively antagonized by the beta(2)-selective antagonists butoxamine and ICI-118,551 with pA(2) values of 6.86 and 8.73, respectively. The pA(2) values of beta-adrenoceptor antagonists (propranolol, bupranolol, atenolol, butoxamine and ICI-118,551) tested against adrenaline were consistent with the values when tested against salbutamol, a beta(2)-selective adrenoceptor agonist. The present findings provide evidence that the relaxant response of the smooth muscle of the guinea-pig trachea to the adrenal medulla hormone, adrenaline, is mainly mediated through beta(2)-adrenoceptors.     

Autoradiographic location of beta-adrenoceptor subtypes in cat colon smooth muscle

In order to localize beta-adrenoceptors 125I-(-)pindolol (IPIN) was used in binding to sections from cat colon. The binding characteristics for IPIN to beta-adrenoceptors on colon sections were estimated by demonstrating reversible binding in the presence of isoprenaline and by steroselective binding to the isomers of propranolol. The binding of IPIN to both beta 1- and beta 2-adrenoceptors was shown by biphasic displacement curves in the presence of the selective beta-adrenoceptor compounds betaxolol, ICI 118.551 and procaterol. The colon sections were found to contain proportions of beta 1-adrenoceptors (30-50%) and beta 2-adrenoceptors (50-70%). In the autoradiographic studies, 100% of the developed grains after exposure of IPIN to the photographic emulsion were displaced by 50 microM of isoprenaline. By microscopic counting at autoradiographic grains, 30-40% of the grains were found in the circular smooth muscle, while 60-70% of the grains were found in the longitudinal smooth muscle. A concentration of 2 nM ICI 118.551 completely displaced all grains in the circular smooth muscle and partly displaced those found in the longitudinal smooth muscle. A high concentration of ICI 118.551 (1 microM) displaced all grains above background from the smooth muscle. It is concluded that the circular smooth muscle only contains beta 2-adrenoceptors, while longitudinal smooth muscle may contain a proportion of beta 1-adrenoceptors. Whether such a location of beta adrenoceptors can be related to the beta 1-adrenoceptor-mediated inhibition of colon motility can not be clarified from these studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide (NO) primarily accounts for endothelium-dependent component of beta-adrenoceptor-activated smooth muscle relaxation of mouse aorta in response to isoprenaline.

Isoprenaline is known to produce vascular relaxation through activation of beta-adrenoceptors. In recent years, beta-adrenoceptor-activated vascular relaxation has been the focus of pharmacological study in terms of both the receptor subtypes and the intracellular signaling mechanisms which trigger smooth muscle mechanical functions. In addition, the possible contribution of the endothelium to beta-adrenoceptor-activated relaxation of vascular beds has provoked considerable discussion, with consensus still to be established. In the present study, we examined the effects of isoprenaline on isolated mouse aortic smooth muscles to determine whether the presence of the endothelium plays a substantial role in the relaxation it produces. A possible role for nitric oxide (NO) as a primary endothelium-derived factor released in response to isoprenaline was also elucidated pharmaco-mechanically. In isolated thoracic and abdominal aortae pre-contracted with phenylephrine (3 x 10(-7)-10(-6) M), isoprenaline elicited relaxation in a concentration-dependent fashion (10(-9)-10(-5) M). In endothelium-denuded preparations, isoprenaline-elicited relaxation was reduced to 40-50% of the response obtained in endothelium-intact preparations. In the preparations treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) M; an NO synthase inhibitor) or 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 10(-5) M; a soluble guanylyl cyclase inhibitor), isoprenaline-elicited relaxation was attenuated almost to the same degree as the response in endothelium-denuded preparations. The degree of endothelium-dependency in isoprenaline-elicited relaxation was largely diminished when treated with propranolol (3 x 10(-6) M). The present findings indicate that isoprenaline substantially relaxes the mouse aorta with both endothelium-dependent and -independent mechanisms. The endothelium-dependent component seems to correspond to about 50% of the isoprenaline-elicited relaxation, and is almost entirely due to endothelium-derived NO. Activation of propranolol (3 x 10(-6) M)-inhibitable beta-adrenoceptors seems to be primarily responsible for the NO-mediated endothelium-dependent pathway in isoprenaline-elicited relaxant response of mouse aorta.     

Autoradiographic location of β-adrenoceptor subtypes in cat colon smooth muscle

In order to localize β-adrenoceptors ¹²⁵I-(—)pindolol (IPIN) was used in binding to sections from cat colon. The binding characteristics for IPIN to β-adrenoceptors on colon sections were estimated by demonstrating reversible binding in the presence of isoprenaline and by steroselective binding to the isomers of propranolol. The binding of IPIN to both β₁-and β₂-adrenoceptors was shown by biphasic displacement curves in the presence of the selective β-adrenoceptor compounds betaxolol, ICI 118.551 and procaterol. The colon sections were found to contain proportions of β₁-adrenoceptors (30–50%) and β₂-adrenoceptors (50–70%). In the autoradiographic studies, 100% of the developed grains after exposure of IPIN to the photographic emulsion were displaced by 50 μm of isoprenaline. By microscopic counting at autoradiographic grains, 30–40% of the grains were found in the circular smooth muscle, while 60–70% of the grains were found in the longitudinal smooth muscle. A concentration of 2 nm ICI 118.551 completely displaced all grains in the circular smooth muscle and partly displaced those found in the longitudinal smooth muscle. A high concentration of ICI 118.551 (1 μm ) displaced all grains above background from the smooth muscle. It is concluded that the circular smooth muscle only contains β₂-adrenoceptors, while longitudinal smooth muscle may contain a proportion of β₁-adrenoceptors. Whether such a location of β-adrenoceptors can be related to the β₁-adrenoceptor-mediated inhibition of colon motility can not be clarified from these studies. However, it seems that β₁-adrenoceptors are located to the longitudinal smooth muscle instead of to the myenteric plexus of the colon.     

Myofilament-based relaxant effect of isoprenaline revealed during work-loop contractions in rat cardiac trabeculae

In cardiac muscle, β-adrenergic stimulation increases contractile force and accelerates relaxation. The relaxant effect is thought to be due primarily to stimulation of Ca²⁺ uptake into the sarcoplasmic reticulum (SR), although changes in myofilament properties may also contribute. The present study investigated the contribution of the myofilaments to the β-adrenergic response in isolated rat cardiac trabeculae undergoing either isometric or work-loop contractions (involving simultaneous force generation and shortening) at different stimulation frequencies (range 0.25-4.5 Hz). SR-dependent effects were eliminated by treatment with ryanodine (1 μM) and cyclopiazonic acid (30 μM). In isometric contractions during SR inhibition, isoprenaline increased the force but did not alter the time course of the twitch. In contrast, in work-loop contractions, the positive inotropic effect was accompanied by a reduced diastolic force between beats, most apparent at higher frequencies (e.g. diastolic stress fell from 58.6 ± 5.5 to 28.8 ± 5.8 mN mm⁻² at 1.5 Hz). This relaxant effect contributed to a β-adrenoceptor-mediated increase in net work and power output at higher frequencies, by reducing the amount of work required to re-lengthen the muscle. Consequently, the frequency for maximum power output increased from 1.1 ± 0.1 to 1.6 ± 0.1 Hz. We conclude that the contribution of myofilament properties to the relaxant effect of β-stimulation may be of greater significance when force and length are changing simultaneously (as occurs in the heart) than during force development under isometric conditions.     

Separate roles for beta2- and beta3-adrenoceptors in memory consolidation

Consolidation of a labile memory which would not normally be stored can be achieved by intracerebral administration of noradrenaline. In a series of experiments using discriminated, one trial passive avoidance learning with the day-old chick, the effect of noradrenaline has been shown to be due to actions at different subtypes of adrenoceptors. The effect of noradrenaline is dose-dependent, with a moderate dose producing memory consolidation. However, higher doses of noradrenaline (0.3-10 nmol/hemisphere) prevent consolidation, an effect not seen with isoprenaline suggesting that these doses stimulate alpha-adrenoceptors. The promotion of memory consolidation by noradrenaline or isoprenaline at low doses was attributable to beta3-adrenoceptors and at medium doses to beta2-adrenoceptors. At higher doses of noradrenaline, there was alpha1-adrenoceptor-mediated inhibition of memory consolidation. Consolidation can also be achieved by administration of either beta2- or beta3-adrenoceptor agonists at specific times after training. Although these two adrenoceptors both promoted memory consolidation, there was a differential action on the stages of memory formation. The dose-response curve to the beta3- and the beta2-agonists was shifted by the appropriate antagonist but not by the antagonist at the other beta-adrenoceptor. Although beta1-adrenoceptors are present in chick brain, they do not seem to have a role in memory formation. These results explain why noradrenaline, acting at different adrenoceptors, can have different effects on memory formation with memory being either consolidated or inhibited depending on the dose. The findings also demonstrate a role in memory formation for beta3-adrenoceptors found in the brain. Agonists acting specifically at beta2- or beta3-adrenoceptors may be of value in diseases involving cognitive impairment.     

MaxiK channel roles in blood vessel relaxations induced by endothelium-derived relaxing factors and their molecular mechanisms.

The endothelium of blood vessels plays a crucial role in the regulation of blood flow by controlling mechanical functions of underlying vascular smooth muscle. The regulation by the endothelium of vascular smooth muscle relaxation and contraction is mainly achieved via the release of vasoactive substances upon stimulation with neurohumoural substances and physical stimuli. Nitric oxide (NO) and prostaglandin I2 (prostacyclin, PGI2) are representative endothelium-derived chemicals that exhibit powerful blood vessel relaxation. NO action involves activation of soluble guanylyl cyclase and PGI2 action is initiated by the stimulation of a cell-surface receptor (IP receptor, IPR) that is coupled with Gs-protein-adenylyl cyclase cascade. Many studies on the mechanisms by which NO and PGI2 elicit blood vessel relaxation have highlighted a role of the large conductance, Ca2+-activated K+ (MaxiK, BKCa) channel in smooth muscle as their common downstream effector. Furthermore, their molecular mechanisms have been unravelled to include new routes different from the conventionally approved intracellular pathways. MaxiK channel might also serve as a target for endothelium-derived hyperpolarizing factor (EDHF), the non-NO, non-PGI2 endothelium-derived relaxing factor in some blood vessels. In this brief article, we review how MaxiK channel serves as an endothelium-vascular smooth muscle transducer to communicate the chemical signals generated in the endothelium to control blood vessel mechanical functions and discuss their molecular mechanisms.     

Effect of Bordetella pertussis Vaccination in Mice and the Isolated Tracheal Response to Isoprenaline

The administration of Bordetella pertussis vaccine to mice has been associated with the development of an impaired beta-adrenoceptor responsiveness and in many respects has resembled human asthma. Trachea (n = 12) were isolated from Swiss-Webster mice 5 days following the intraperitoneal administration of 2 x 10(9) B. pertussis organisms. The tracheal smooth muscle response to carbachol was measured and compared with that found in trachea from unvaccinated mice (n = 15). The contractile response was similar in both groups. The tracheal smooth muscle relaxant effects of isoproterenol were measured in these two groups. The EC50 value for isoprenaline (6.5 x 10(-7) M) in trachea from B. pertussis treated mice was significantly (P < 0.05) greater than that noted in the control animals (2.3 x 10(-7) M). These studies demonstrated that in tracheal smooth muscle isolated from B. pertussis vaccinated mice, the relaxant effects of isoprenaline are impaired.     

The bronchorelaxant effect of helicidine, a Helix pomatia extract, interferes with prostaglandin E2

Helicidine is a biological extract prepared from the snail Helix pomatia L. and used in man as an anti-tussive agent. However, its mechanisms of action are not fully defined. In this study, we have investigated a possible relaxant effect of helicidine on guinea-pig airway smooth muscle and evaluated the role of prostanoids and airway epithelium in this relaxation. H. pomatia extract (0.001-1 mg/ml) induced a dose-dependent relaxation of guinea-pig trachea pre-contracted with histamine both in the presence and absence of tracheal epithelium. No significant difference in dose-dependency or magnitude of the relaxation was observed between tracheal segments with or without epithelium (maximal relaxant response of 35 +/- 7 and 25 +/- 7.5%, respectively). Relaxation of the trachea induced by H. pomatia extract (0.001-1 mg/ml) was inhibited by pre-treatment with the cyclooxygenase inhibitor, indomethacin, both in the presence or absence of tracheal epithelium. H. pomatia extract (1 mg/ml) induced a marked and significant increase in prostaglandin E2 release in tracheal segments with and without epithelium. These results indicate that helicidine possesses a broncho-relaxant activity which is independent of epithelium integrity and which is partly mediated by the release of the relaxant prostanoid, prostaglandin E2. The origin of prostaglandin E2 production in the airways remains to be defined.     

KCNMB1 regulates surface expression of a voltage and Ca2+-activated K+ channel via endocytic trafficking signals

Voltage-dependent and calcium-activated K(+) (MaxiK, BK) channels are ubiquitously expressed and have various physiological roles including regulation of neurotransmitter release and smooth muscle tone. Coexpression of the pore-forming alpha (hSlo) subunit of MaxiK channels with a regulatory beta1 subunit (KCNMB1) produces noninactivating currents that are distinguished by high voltage/Ca(2+) sensitivities and altered pharmacology [McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14:645-650; Wallner M, Meera P, Ottolia M, Kaczorowski G, Latorre R, Garcia ML, Stefani E, Toro L (1995) Characterization of and modulation by a beta-subunit of a human maxi K(Ca) channel cloned from myometrium. Receptors Channels 3:185-199]. We now show that beta1 can regulate hSlo traffic as well, resulting in decreased hSlo surface expression. beta1 subunit expressed alone is able to reach the plasma membrane; in addition, it exhibits a distinct intracellular punctated pattern that colocalizes with an endosomal marker. Coexpressing beta1 subunit with hSlo, switches hSlo's rather diffuse intracellular expression to a punctate cytoplasmic localization that overlaps beta1 expression. Furthermore, coexpressed beta1 subunit reduces steady-state hSlo surface expression. Site-directed mutagenesis underscores a role of a putative endocytic signal at the beta1 C-terminus in the control of hSlo surface expression. We propose that aside from its well-established role as regulator of hSlo electrical activity, beta1 can regulate hSlo expression levels by means of an endocytic mechanism. This highlights a new beta1 subunit feature that regulates hSlo channels by a trafficking mechanism.     

MaxiK channel and cell signalling

The large-conductance Ca²⁺- and voltage-activated K⁺ (MaxiK, BK, BK_Ca, Slo1, K_Ca1.1) channel role in cell signalling is becoming apparent as we learn how the channel interacts with a multiplicity of proteins not only at the plasma membrane but also in intracellular organelles including the endoplasmic reticulum, nucleus, and mitochondria. In this review, we focus on the interactions of MaxiK channels with seven-transmembrane G protein-coupled receptors and discuss information suggesting that, the channel big C-terminus may act as the nucleus of signalling molecules including kinases relevant for cell death and survival. Increasing evidence indicates that the channel is able to associate with a variety of receptors including β-adrenergic receptors, G protein-coupled estrogen receptors, acetylcholine receptors, thromboxane A2 receptors, and angiotensin II receptors, which highlights the varied functions that the channel has (or may have) not only in regulating contraction/relaxation of muscle cells or neurotransmission in the brain but also in cell metabolism, proliferation, migration, and gene expression. In line with this view, MaxiK channels have been implicated in obesity and in brain, prostate, and mammary cancers. A better understanding on the molecular mechanisms underlying or triggered by MaxiK channel abnormalities like overexpression in certain cancers may lead to new therapeutics to prevent devastating diseases.     

Mechanisms of the relaxant and contractile responses to bradykinin in rat duodenum

The signal pathway for bradykinin-induced relaxation followed by contraction in the isolated rat duodenum was investigated by comparing the effect of blocking agents on the response to bradykinin and acetylcholine. The phospholipase C inhibitor U-73122 inhibited the relaxation induced by bradykinin, but had no effect on the contraction to either bradykinin or acetylcholine. The same response pattern was observed when the tissues were pre-treated with thapsigargin, a selective inhibitor of microsomal Ca2+ pumps. An inhibitor of non-voltage-dependent Ca2+ influx, SK&F 96365, inhibited the relaxant response to bradykinin and the contraction induced by acetylcholine, but not the contraction induced by bradykinin. In Ca2+-free Krebs-Henseleit buffer, the tissues failed to respond when they were exposed to either bradykinin or acetylcholine. When the tissues were partly depolarized (30 mM KCI), both bradykinin and acetylcholine induced contraction, while the relaxant response to bradykinin was almost completely abolished. Apamin (an antagonist of low-conductance calcium-activated K+ channel) together with charybdotoxin (CTX, an antagonist of large-conductance calcium-activated K+ channel) and CTX alone inhibited the relaxant but not the contractile response to bradykinin. We conclude that the biphasic response in isolated rat duodenum to bradykinin involves two distinct pathways. We propose that the relaxant component is induced indirectly via inositol-mediated increase in cytosolic Ca2+ in non-muscle cells with subsequent signals to the smooth muscle cells, whereas the contractile response is induced by direct effect on the smooth muscle cells.     

乙酰胆碱和组织胺对豚鼠离体气管条张力的影响及机制分析

用自制的多个“工”形相连的豚鼠气管条,观察乙酰胆碱、组织胺和异丙基肾上腺素对肌张力的影响,探讨钙激活钾(KC_a)通道功能的变化。结果显示:1.乙酰胆碱和组织胺共同作用时,平滑肌张力升高幅度大于乙酰胆碱和组织胺单独作用,小于两者单独作用之和。2.异丙基肾上腺素引起乙酰胆碱或组织胺所致平滑肌张力下降,甚至低于基础张力,乙酰胆碱和组织胺共同作用时,异丙基肾上腺素也可解痉。结果提示:中等浓度乙酰胆碱和组织胺对增加气管平滑肌张力有协同作用。异丙基肾上腺素引起乙酰胆碱或组织胺所致平滑肌张力下降,表明乙酰胆碱和组织胺引起气管平滑肌痉挛时,KC_a通道处于关闭或大部分关闭状态。     

The beta3-adrenoceptor-mediated relaxation induced by epinephrine in guinea pig taenia caecum.

The mechanisms of the beta-adrenoceptor mediated relaxation induced by epinephrine in guinea pig taenia caecum were examined. The relaxant response to epinephrine was unaffected by propranolol (approximately 10(-5) M) or phentolamine (approximately 10(-5) M). The response to epinephrine was antagonized in a concentration dependent manner by bupranolol, and Schild plot of the data revealed the pA2 value of 5.87. Epinephrine significantly increased cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP level that was elicited by epinephrine, whereas propranolol (10(-5) M) produced no effect. These results suggest that the relaxant response to epinephrine in the guinea pig taenai caecum is mainly mediated by beta3-adrenoceptors.     

Relaxation mechanisms induced by stimulation of nerves and by nitric oxide in sheep urethral muscle.

Isolated transverse and longitudinally oriented preparations of sheep urethra precontracted with noradrenaline responded to electrical field stimulation (EFS) with stimulus-dependent non-adrenergic, non-cholinergic (NANC) relaxations. Exogenous nitric oxide (NO) (acidified NaNO2), S-nitroso-L-cysteine (NC), sodium nitroprusside (SNP), 8-Br-cGMP, dibutyryl-cAMP, forskolin and isoprenaline each relaxed precontracted transverse urethral preparations in a concentration-dependent manner in order of protency: NC > forskolin > isoprenaline = SNP > NO > 8-Br-cGMP = dibutyryl-cAMP. Longitudinally oriented preparations responded to NO and NC with concentration-dependent relaxation, no different from that observed in transverse strips. Methylene blue (MB) and oxyhaemoglobin (HbO2) each shifted the concentration-response curve for NO to the right without affecting EFS-induced relaxation. Similarly, concentration-dependent responses to NC were not affected by MB. The inhibition of relaxation to NO by MB was prevented by superoxide dismutase, suggesting the inhibition was caused by extracellular generation of superoxide anions. EFS-induced relaxation was accompanied by elevation of cGMP. However, for the same level of relaxation, exogenous NO and NC induced 15- and 23-times higher increases in cGMP values, respectively, than EFS. cAMP levels were not affected by EFS- or NO-induced relaxation, although a large increase accompanied relaxation induced by forskolin. Forskolin also increased cGMP content. Pretreatment with MB reduced basal levels of cGMP and inhibited both relaxation and rise in cGMP levels induced by NO. SNP-elicited relaxant responses, in the presence of MB, were accompanied by an accumulation of cGMP; cAMP levels were unaffected. MB reduced cGMP levels induced by NC, while the relaxant response was unchanged. In urethral preparations prelabelled with [3H]myoinositol, exposure to NA caused an accumulation of [3H]inositol phosphates, which was unaffected by pretreatment with 8-Br-cGMP or dibutyryl-cAMP. EFS failed to induce a relaxant response in excess [K+]o-contracted preparations, while relaxation with exogenous NO was unaffected. Ouabain abolished EFS-induced relaxation and reduced responses to NO. Neither TEA nor glibenclamide affected relaxation to either EFS or NO. Relaxation elicited by SNP was not accompanied by any change in cGMP or cAMP levels, and was unaffected by MB, HbO2, K+ channel blockers (TEA and glibenclamide), ouabain or high [K+]o solution. This suggested that relaxation was caused by a mechanism independent of NO generation. A dense network of NADPH diaphorase-positive fibres associated with both the circular and longitudinal smooth muscle layers of sheep urethra was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Small- and Intermediate-Conductance Calcium-Activated K+ Channels Provide Different Facets of Endothelium-Dependent Hyperpolarization in Rat Mesenteric Artery

Activation of both small-conductance (SK_Ca) and intermediate-conductance (IK_Ca) Ca²⁺-activated K⁺ channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K⁺ channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of a nitric oxide (NO) synthase inhibitor, ACh evoked endothelium and concentration-dependent smooth muscle hyperpolarization, increasing the resting potential (approx. −53 mV) by around 20 mV at 3 μ m . Similar hyperpolarization was evoked with cyclopiazonic acid (10 μ m , an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1-EBIO (300 μ m , an IK_Ca activator) only increased the potential by a few millivolts. Hyperpolarization in response to either ACh or CPA was abolished with apamin (50 n m , an SK_Ca blocker) but was unaltered by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (1 μ m TRAM-34, an IK_Ca blocker). During depolarization and contraction in response to phenylephrine (PE), ACh still increased the membrane potential to around −70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential ( circa −58 mV). TRAM-34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh-evoked hyperpolarization was completely abolished. These data suggest that true endothelium-dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SK_Ca channels, whereas IK_Ca channels play an important role during the ACh-mediated repolarization phase only observed following depolarization.