Excitation-contraction coupling in canine tracheal smooth muscle cells.
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3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Biomedical subjects
E E Daniel
Publications and source records attributed to E E Daniel.
Participation of protein kinase C in endothelin-1-induced contraction in rat aorta: studies with a new tool, calphostin C.
1. Calphostin C at 10(-6) M was shown to be selective and highly effective in inhibiting contractile responses of rat aortae to 12-o-tetradecanoylphorbol-13-acetate, while it had no effect on contractile responses to elevated KCl. 2. In the rat aorta, endothelin-1 (ET-1) developed a sustained tonic contraction dose-dependently in both normal Ca(2+)-containing Krebs and Ca(2+)-free Krebs containing 1 mM EGTA. Calphostin C (10(-6) M), a selective protein kinase C inhibitor, antagonized the maximal tensions for cumulative addition of 10(-8) M ET-1 by 13.2% in Ca(2+)-containing medium and 25.8% in Ca(2+)-free Krebs containing 1 mM EGTA. 3. In both Ca(2+)-containing medium and Ca(2+)-free Krebs containing 1 mM EGTA, precontraction with 10(-8) M ET-1 had no effects on the contractile response to subsequently added 10(-6) M 12-o-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. 4. In Ca(2+)-free Krebs containing 1 mM EGTA, precontraction with 10(-6) M TPA potentiated the contractile response to subsequently added 10(-8) M ET-1, whereas this potentiation was abolished by pretreatment with 10(-6) M calphostin C. The mechanism of the TPA-induced potentiating effect remains to be determined. 5. These results suggest that the participation of protein kinase C in the 10(-8) M ET-1-induced contraction may be 13.2% and 25.8% in the presence and absence of extracellular Ca2+, respectively, and that mechanisms other than protein kinase C may be predominantly responsible for ET-1-induced tonic contraction.
Animals↗
K(+)-channel blockers do not decrease acetylcholine depolarizations in canine trachealis.
Using the double sucrose gap, we have examined the role of K+ channels in the cholinergic depolarizations in response to field stimulation and acetylcholine (Ach) in canine trachealis. Acetylcholine-like depolarization per se decreased electrotonic potentials from hyperpolarizing currents. The net effect of acetylcholine (10(-6) M) depolarization on membrane conductance was a small increase after the depolarization was compensated by current clamp. Reversal potentials for acetylcholine depolarization and for the excitatory junction potential (EJP) were determined by extrapolation to be 20-30 mV positive to the resting potential, previously shown to be approximately -55 mV. They were shifted positively by tetraethylammonium ion (TEA) at 20 mM or Ba2+ at 1 mM. TEA or Ba2+ initially depolarized the membrane and increased membrane resistance. Repolarization of the membrane restored any reductions in EJP amplitudes associated with depolarization. After 15 min, the membrane potential partially repolarized, and acetylcholine-induced depolarization and contractions were then increased by TEA. 4-Aminopyridine depolarized the membrane but decreased membrane resistance. Apamin (10(-6) M), charybdotoxin (10(-7) M), and glybenclamide (10(-5) M) each failed to significantly depolarize membranes, increase membrane resistance, or reduce EJP amplitudes or depolarization to 10(-6) M Ach. Glybenclamide reduced depolarizations to added acetylcholine slightly. TEA occasionally reduced the EJP markedly, but this was shown to be most likely a prejunctional effect mediated by norepinephrine release. TEA alone among K(+)-channel blockers slowed the onset and the time courses of the EJP as well as the acetylcholine-induced depolarization. K(+)-channel closure cannot be a complete explanation of acetylcholine-induced membrane effects on this tissue. Acetylcholine must have increased the conductance of an ion with a reversal potential positive to the resting potential in addition to any effect to close K+ channels.
Acetylcholine↗
Excitability of canine colon circular muscle disconnected from the network of interstitial cells of Cajal.
The 6 cpm omnipresent slow waves recorded in the circular muscle (CM) layer of canine colon are generated at the submucosal surface of the CM layer. After removal of the submucosal network of interstitial cells of Cajal (ICC), 66% of the CM preparations (25 of 38) were quiescent in Krebs solution. In the presence of carbachol, seven of nine of these spontaneously quiescent CM preparations demonstrated slow wave-like activity with mean frequency, duration and amplitude of 5.9 +/- 0.4 cpm, 2.8 +/- 0.5 s, and 0.8 +/- 0.2 mV, respectively. Similar slow wave-like activities were induced by TEA (seven out of eight quiescent CM preparations) with frequency, duration and amplitude of 6.1 +/- 0.2 cpm, 2.7 +/- 0.5 s, and 1.0 +/- 0.2 mV, respectively, and by BaCl2 (eight of eight quiescent CM preparations) with frequency, duration, and amplitude of 6.3 +/- 0.3 cpm, 1.8 +/- 0.2 s, and 0.5 +/- 0.1 mV, respectively. All the induced activities were abolished in the presence of 1 microM D600. CM preparations with the submucosal ICC network intact (ICC-CM) showed slow wave activity in Krebs solution at a frequency of 6.2 +/- 0.2 cpm, a duration of 3.6 +/- 0.2 s, and an amplitude of 1.0 +/- 0.1 mV (n = 22). When ICC-CM preparations were stimulated by BaCl2, carbachol, or TEA, the slow wave frequency did not change significantly, but the duration increased as well as the amplitude. In the presence of D600, the upstroke of slow waves remained and the frequency was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Developments in the study of smooth muscle research: reflections on 42 years.
This review of smooth muscle research in my laboratory covers over 40 years. It focuses on research on the control of myometrium, blood vessels, gastrointestinal muscle, and airway muscle and highlights the major findings and those who made them. My goal is to show how science can be rewarding both in terms of findings and in terms of the people who make them.
Animals↗
Effects of inflammatory mediators on canine airway neuromuscular function.
Inflammatory mediators can both enhance or inhibit canine airway reactivity. PGE2 and PGI2 in general are inhibitory, interfering with release of acetylcholine and with responses to bronchoconstrictors. These prostaglandins may be more effective against agonists that open voltage-dependent Ca2+ channels to induce Ca2+ influx and contraction compared with those agonists that release internal Ca2+. Other mediators are excitatory: histamine, PGD2, thromboxane A2 (TxA2), and leukotrienes (LT) C4, D4, and E4. In canine airway only histamine and TxA2 have effects in the absence of indomethacin, i.e., in the presence of the large amounts of PGE2 and PGI2 produced in vitro LTs are ineffective. Effects of TxA2 and histamine may be potentiated if the synthesis of these inhibitory PGs is inhibited. Whether histamine or TxA2 normally promote synthesis and release of PGE2 and PGI2 in a kind of homeostasis remains to be explored. It is also unclear whether pre- as well as post-junctional TxA2 receptors exist and have different pharmacological sensitivities to antagonists. LTC4 and LTD4 also constrict canine bronchi but only when PGE2 and PGI2 synthesis is blocked and, again, whether this is a result of LT-induced release of inhibitory mediators is unknown. The concept that airway responsiveness can be caused by turning off PGE2 and PGI2 production and turning on TxA2 or LT production (or unmasking their actions) needs further exploration. Our recent data suggest that such a mechanism may explain ozone-induced responsiveness in dogs.
Acetylcholine↗
A mediator derived from arginine mediates inhibitory junction potentials and relaxations in lower esophageal sphincter: an independent role for vasoactive intestinal peptide.
This study provides mechanical and electrophysiological evidence to show that a metabolite of arginine, not vasoactive intestinal peptide (VIP), is the putative nonadrenergic noncholinergic (NANC) inhibitory mediator in canine and opossum lower esophageal sphincters (LES). Relaxations of spontaneous active tension by electrical field stimulation (FS) at parameters that induced tetrodotoxin (TTX)-sensitive responses were abolished by L-N omega-arginine methyl ester (L-NAME) at 10(-4) M and restored by L-arginine (10(-3) M) but not D-arginine (10(-3) M). TTX-insensitive relaxations to 5-ms pulses were unaffected by L-NAME, L- or D-arginine. VIP (10(-6) M) caused maximum relaxations of basal tension in both the opossum and canine LES. However these relaxations, unlike those from FS were unaffected by L-NAME. Methylene blue (5 x 10(-5)M) increased basal tension of the LES in each species, and did not inhibit the relaxation to FS or VIP, but often increased the amplitudes of these responses due to the increase in basal tension. In parallel experiments NANC inhibition of body circular muscle from opossum esophagus was abolished by methylene blue. Electrophysiological studies using micro-electrodes revealed that NANC inhibition was associated with inhibitory junction potentials in the canine LES. These were inhibited by L-NAME and restored by L-arginine but not D-arginine. In contrast, 10(-6) M VIP in canine LES did not induce any change in membrane potential during a 20-min superfusion. Sodium nitroprusside also hyperpolarized sphincteric muscle and its effects were not affected by L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Inhibitory and excitatory mechanisms of neurotensin action in canine intestinal circular muscle in vitro.
The effect of neurotensin on canine ileal circular muscle devoid of myenteric plexus was investigated using single and double sucrose gap techniques. Similar results were obtained with microelectrode techniques. Neurotensin caused a temperature-sensitive and dose-dependent biphasic response, an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase, usually consisting of spike discharge and tonic and phasic contractions, for which depolarization was not required. Neither response was affected by tetrodotoxin, phentolamine, propranolol, or atropine. The hyperpolarization was associated with decreased membrane resistance, blocked by 10(-7) M apamin, and converted to tonic depolarization by apamin (10(-6) M). Tachyphylaxis to neurotensin occurred when the stimulation interval was less than 20 min. After Ca2+ depletion, depolarization was observed instead of the hyperpolarization; this depolarization was not affected by nitrendipine and was gradually abolished with repetitive stimulation at 20-min intervals. When Ca2+ was present, nifedipine did not alter the hyperpolarizing phase of the response but inhibited spiking and blocked all contractions. The excitatory phase of the response was enhanced by Bay K-8644. Neuromedin N elicited a response identical with that of neurotensin. The responses of the two peptides were completely cross tachyphylactic. Inhibitory junction potentials were not affected by neurotensin tachyphylaxis. It is concluded that neurotensin and neuromedin N activate apamin-sensitive, calcium-dependent potassium channels in circular muscle, causing membrane hyperpolarization and inhibition of muscle contraction. Release of intracellular calcium is involved in the activation of these potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Nitric oxide as a putative nonadrenergic noncholinergic inhibitory transmitter in the canine pylorus in vivo.
Antropyloroduodenal motility was recorded in seven anesthetized dogs to assess the role of nitric oxide and L-arginine metabolites in nonadrenergic noncholinergic (NANC) mediation of pyloric relaxation. Pyloric activity induced by duodenal field stimulation was inhibited by antral field stimulation and electrical vagal stimulation. Intra-arterial NG-L-arginine-methyl-ester (L-NAME) reduced the inhibition from antral or vagal stimulation (P less than 0.05). Intravenous infusion of L-NAME also blocked the inhibitory effect of vagal and antral stimulation but left the tetrodotoxin-insensitive action of intra-arterial vasoactive intestinal peptide (VIP) and sodium nitroprusside unchanged. L-Arginine reversed the effect of L-NAME whereas D-arginine did not. L-NAME enhanced pyloric contractions to intra-arterial acetylcholine. The NANC inhibition of the substance P-stimulated pyloric response in vitro was blocked by L-NAME and reversed by addition of L-arginine. Sodium nitroprusside was effective as a relaxant in vitro but VIP was not. These data suggest that metabolites of L-arginine mediate neural inhibition of canine pyloric motor activity.
Animals↗
Chloride-mediated inhibitory junction potentials in opossum esophageal circular smooth muscle.
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Animals↗
Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms.
Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 microM) in Ca(2+)-free medium containing 50 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 microM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 microM), and L-type Ca2+ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 microM), an L-type Ca2+ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KCl (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 microM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2+ pump for Ca2+ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca(2+)-free medium may reflect compartmentalization of Ca2+ storage related to the different routes of refilling.
Animals↗
Effect of an anti-Mo1 MAb on ozone-induced airway inflammation and airway hyperresponsiveness in dogs.
Ozone inhalation causes neutrophil migration into the airway and airway hyperresponsiveness in dogs. The leukocyte adhesion molecule Mo1 (CD11b/CD18) is a heterodimeric glycoprotein the expression of which is necessary for neutrophil adhesion to endothelium. To evaluate the contribution of Mo1 to ozone-induced neutrophil influx and airway hyperresponsiveness, six dogs were treated intravenously with an Anti-Mo1 monoclonal antibody (3.75 mg/kg in normal saline) that binds to both human and canine Mo1, or the diluent alone, 1.5 h before inhaling ozone (3 ppm for 30 min), or dry air. Airway responses to doubling doses of inhaled acetylcholine (ACh) were measured before and after inhalation of ozone. Neutrophil influx was assessed by bronchoalveolar lavage (BAL) performed after the second ACh inhalation. Treatment with anti-Mo1 prevented the ozone-induced influx of neutrophils into BAL. After diluent and inhaled dry air, the neutrophil count in BAL was 1.49 +/- 1.26 (SE) x 10(4) (5.0% of total cells). After diluent and inhaled ozone, the neutrophil count increased to 7.27 +/- 3.22 (SE) x 10(4) (22.6% of total cells) (P < 0.05). After anti-Mo1 and inhaled ozone, the neutrophil count was 1.48 +/- 0.62 (SE) x 10(4) (8.5% of total cells). Treatment with anti-Mo1 also significantly reduced the number of eosinophils in BAL after ozone. Ozone-induced ACh airway hyperresponsiveness was not prevented by treatment with anti-Mo1. These results indicate that expression of Mo1 is necessary for ozone-induced neutrophil migration into the airway lumen.
Acetylcholine↗
Amplification of alpha adrenergic vasoconstriction in canine isolated mesenteric artery and vein.
The interactions between UK-14304 and other vasoconstrictor agents were investigated using isolated canine mesenteric vascular rings mounted in tissue baths for the measurement of isometric contraction. In the mesenteric artery, exposure to UK-14304 caused a small contraction, producing 8% of the KCl maximal response. In the presence of either 20 mM KCl or 10(-9) M endothelin-1, which had small contractile effects, UK-14304 produced a biphasic concentration-response curve; 10(-7) M rauwolscine inhibited the responses to low concentrations of UK-14304 and 10(-7) M prazosin blocked the responses to high concentrations of UK-14304. In the presence of 10(-8) M Bay K 8644, UK-14304 elicited a monophasic concentration-dependent contraction that was antagonized by 10(-7) M prazosin, not by 10(-7) M rauwolscine. In the mesenteric vein, UK-14304 elicited concentration-dependent contractions, producing 63% of the KCl maximal response. The lower part of the biphasic concentration-response curve was inhibited by 10(-7) M rauwolscine and the upper part of the curve was antagonized by 10(-7) M prazosin. The presence in the medium of 20 mM KCl, 10(-11) M endothelin-1 or 10(-9) M Bay K 8644, which increased resting tension less than 10% of the KCl maximal response, markedly enhanced the responses to UK-14304 primarily at concentrations lower than 10(-6) M. The enhancement of responses were prazosin (10(-7) M)-resistant and rauwolscine (10(-7) M)-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Prejunctional muscarinic receptors in the deep muscular plexus of canine ileum: comparison with smooth muscle receptors.
Prejunctional muscarinic receptors from the deep muscular plexus of canine ileum were studied, and their properties were compared with those of the postjunctional receptors of the circular smooth muscle. In the purified synaptosomal fraction (a fraction containing primarily the axonal varicosities of deep muscular plexus), the muscarinic ligand N-[3H]methylscopolamine labeled an apparently homogenous population of receptors (nH = 1) with a Kd of 2.7 nM and a Bmax of 195 +/- 44 fmol/mg protein (mean +/- S.D., n = 4). These receptors showed a high affinity for the M3/M1-selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (pKi = 7.41); in contrast, the pKi values of pirenzepine (5.60), methoctramine (5.65) and AF-DX 116 (5.21) implied little selectivity for these subtypes. The binding properties of muscarinic receptors in the synaptosomal fraction were different from the binding properties of muscarinic receptors in the purified circular smooth muscle plasma membranes. Most notably, the circular smooth muscle receptors had significantly lower affinity for N-[3H]methylscopolamine (Kd = 16 nM) with a Bmax value of 2088 +/- 276 fmol/mg. The affinities of the M2 subtype-selective muscarinic antagonists methoctramine and AF-DX 116 were similar in both membrane preparations. The receptor population associated with the deep muscular plexus synaptosomal fraction was linked to the inhibition of adenylate cyclase activity, as demonstrated by a concentration-dependent, atropine-sensitive inhibition of the forskolin-stimulated enzyme in the presence of muscarinic agonists carbachol and oxotremorine. Based on the pharmacological observations presented here, the prejunctional muscarinic receptors in the axonal varicosities of deep muscular plexus are different from the postjunctional receptors present in the circular smooth muscle.
Adenylyl Cyclases↗
Vascular effects of tetramethylpyrazine: direct interaction with smooth muscle alpha-adrenoceptors.
The interaction of tetramethylpyrazine, a vasoactive ingredient of a Chinese traditional medicinal plant, with the vascular muscle alpha 1-adrenoceptors was investigated by a direct radioligand binding technique using [3H]prazosin and vascular smooth muscle microsomes isolated from dog aorta and mesenteric artery. Tetramethylpyrazine inhibited the binding of [3H]prazosin to vascular muscle membranes in a concentration-dependent manner at a suboptimal concentration of prazosin. Scatchard analysis of the effect of tetramethylpyrazine on the saturation profile of [3H]prazosin binding to vascular muscle microsomes of either arterial muscle indicated a substantial increase of Kd values (the affinity for prazosin) without a change in Bmax (maximal binding sites for prazosin). Thus, the present results provide supporting evidence that the inhibitory effect of tetramethylpyrazine on the vasoconstriction of dog mesenteric artery induced by phenylephrine in the earlier studies may be, at least, in part due to a direct action at the recognition sites of alpha 1-adrenoceptors. Amiloride and amiloride-related compounds, which shares a common pyrazine ring structure with tetramethylpyrazine and other related derivatives, also inhibits the binding of [3H]prazosin to aortic muscle microsomal membranes. Functional studies of dog saphenous vein also indicated that both tetramethylpyrazine and its ethyl derivatives inhibited the responses induced by phenylephrine and B-HT 920 in the presence and absence of extracellular Ca2+. Together with our earlier findings that amiloride also inhibits [3H]prazosin and [3H]rauwolscine binding to vascular muscle alpha 1- and alpha 2-adrenoceptors, the present radioligand binding study in canine arteries and functional study in saphenous veins suggest that the above compounds containing the pyrazine nucleus indeed interacted at the alpha-adrenoceptor sites.
Adrenergic alpha-Agonists↗
Sensory nerves of the intestines: role in control of pyloric region of dogs.
The canine pylorus is the target for a large array of extrinsic and intrinsic nerve-produced effects. Some of them have intermediate nicotinic synapses. In all cases observed so far the final nerve terminals mediating excitation are cholinergic, but those mediating inhibition are both adrenergic and non-adrenergic non-cholinergic. The role of the dense peptidergic innervation is unclear especially in view of the observation that this muscle and its nerves contain receptors to all of those studied (VIP, PHI, GAL, SP, NKA, NKB, CCK, and others. The pylorus is also the target of an important reflex initiated by acid in the duodenal lumen. Our preliminary work suggests that this reflex is initiated by acid induced release of 5HT from the EC cells to act on 5HT receptors on sensory nerves which activate the ascending chain of cholinergic nerves in the myenteric plexus. This same stimulus may also activate other vagal sensory nerves.
Animals↗
Galanin inhibition of vasoactive intestinal polypeptide release and circular muscle motility in the isolated perfused canine ileum.
The role of porcine galanin, infused arterially into isolated perfused canine ileal segments, in modulating the tonically elevated neural release of vasoactive intestinal polypeptide and possible concomitant motor actions dependent on vasoactive intestinal polypeptide modulation was studied. Galanin infusions (9-minute) inhibited vasoactive intestinal polypeptide release in a concentration-dependent manner (maximum during minutes 8-10) irrespective of the absence (quiescence) or presence of phasic circular muscle contractions induced by local electrical field stimulation of nerves. During quiescence, galanin induced phasic contractions in four of five segments beginning in the 8th minute of the infusion. During stimulated contractions, galanin inhibited phasic motor activity within 2 minutes of initiation of the infusion; this inhibition may result from direct smooth previously reported muscle inhibition. Thus galanin may inhibit both neural release of vasoactive intestinal polypeptide and circular muscle motility directly. The delayed period of phasic activity initiated by galanin during quiescence may be related to inhibition of vasoactive intestinal polypeptide release, freeing the muscle from tonic inhibition by vasoactive intestinal polypeptide. Because galanin and vasoactive intestinal polypeptide are colocalized in some enteric nerves, galanin may regulate vasoactive intestinal polypeptide release by negative feedback.
Animals↗
Cromakalim and K+ channels in canine trachealis.
The effects of cromakalim and glibenclamide on membrane properties and responses to acetylcholine of canine trachea were studied in the double sucrose gap to evaluate the presence and function of ATP-sensitive K+ channels. Cromakalim produced a concentration-dependent hyperpolarization of muscle membrane potential which at maximum brought the membrane potential near the potassium equilibrium potential. Current clamping by hyperpolarizing current to this equilibrium potential abolished the hyperpolarization but not the membrane resistance decrease to cromakalim. Glibenclamide had no effect on resting membrane properties but reduced or abolished effects of cromakalim. Another K+ channel antagonist, tetraethylammonium at 20 mM, also reduced the effects of cromakalim, but 4-aminopyridine (5 mM), Ba2+ (1 mM), and apamin (10(-6) M) had no antagonistic effect. The EJP produced on stimulation of cholinergic nerves sometimes increased just after cromakalim-induced hyperpolarization, but within 5-10 min as membrane resistance dramatically fell it was reduced, as was the depolarization to infused acetylcholine. Initially the reduction in EJP amplitude could be partially overcome by applying hyperpolarizing currents or by applying a second field stimulation; later the EJP was reduced further and was unaffected by these procedures. Even when depolarization to acetylcholine was markedly reduced, the contraction was not. Glibenclamide had no effects alone but antagonized all the effects of cromakalim. These results suggest that ATP-sensitive cromakalim activated K+ channels are present in canine trachea but are usually closed during resting conditions under our experimental conditions. When they are opened by cromakalim, they hyperpolarize to near EK, markedly decrease membrane resistance and reduce the depolarization response to acetylcholine, probably by short circuiting the acetylcholine-induced current.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcholine↗