Electromechanical effects of tetraethylammonium and K+ on histamine-induced contraction in pig isolated tracheal smooth muscle

The effect of tetraethylammonium (TEA) and K+ on contractions to histamine and acetylcholine have been compared in the pig isolated trachea using organ bath and sucrose-gap techniques. Histamine elicited weak contractions, compared with acetylcholine; however, these contractions were markedly potentiated by pretreatment with TEA (10 mM) or by raising the external KCl concentration to 30-50 mM. Neither TEA nor K+ increased the sustained depolarization evoked by histamine (or acetylcholine) although oscillatory depolarizations were often observed in the presence of TEA. Verapamil and a zero Ca2+ Krebs solution reduced contractions to histamine and reduced or abolished the effect of TEA and K+ on histamine-induced contractions. The results unmask different mechanisms of contraction for histamine and acetylcholine. Histamine-induced tone appears to be linked with mechanisms sensitive to TEA and high K+, possibly involving increased translocation of Ca2+ across the plasma membrane.     

Mechanisms of vasodilation induced by NKH477, a water-soluble forskolin derivative, in smooth muscle of the porcine coronary artery.

To study the mechanism of vasodilation induced by 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative, its effects on the acetylcholine (ACh)-induced contraction of muscle strips of porcine coronary artery were examined. [Ca2+]i, isometric force, and cellular concentrations of cAMP and inositol 1,4,5-trisphosphate were measured. NKH477 (0.1-1.0 microM), isoproterenol (0.01-0.1 microM), or forskolin (0.1-1.0 microM) increased cAMP and attenuated the contraction induced by 128 mM K+ or 10 microM ACh in a concentration-dependent manner. These agents, at concentrations up to 0.3 microM, did not change the amount of cGMP. NKH477 (0.1 microM) attenuated the contraction induced by 128 mM K+ without corresponding changes in the evoked [Ca2+]i responses. ACh (10 microM) produced a large phasic increase followed by a small tonic increase in [Ca2+]i and produced a sustained contraction. The ACh-induced phasic increase in [Ca2+]i, but not the tonic increase, disappeared after application of 0.1 microM ionomycin. NKH477 (0.1 microM) attenuated both the increase in [Ca2+]i and the force induced by 10 microM ACh in muscle strips that were not treated with ionomycin and inhibited the ACh-induced contraction without corresponding changes in [Ca2+]i in ionomycin-treated muscle strips. These results suggest that NKH477 inhibits ACh-induced Ca2+ mobilization through its action on ionomycin-sensitive storage sites. In ionomycin-treated and 128 mM K(+)-treated muscle strips, 0.1 microM NKH477 shifted the [Ca2+]i-force relation to the right in the presence or absence of 10 microM ACh. In beta-escin-skinned smooth muscle strips, 0.1 microM NKH477 shifted the pCa-force relation to the right but had no effects on Ca(2+)-independent contraction. We conclude that in smooth muscle of porcine coronary artery, NKH477 inhibits ACh-induced contraction by both attenuating ACh-induced Ca2+ mobilization and reducing the sensitivity of the contractile machinery to Ca2+, possibly by activating cAMP-dependent mechanisms.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.     

Effects on the rabbit coronary artery of LP-805, a new type of releaser of endothelium-derived relaxing factor and a K+ channel opener.

In the rabbit epicardial coronary artery, 8-tert-butyl-6,7-dihydropyrolo[3,2-e]5-methylpyrazolo [1,5-a]pyrimidine-3-carbonitrile (LP-805, greater than 0.1 microM) hyperpolarized the muscle membrane in both proximal (diameter, 1-1.2 mm) and distal (diameter, 0.1-0.2 mm) regions of intact (+E) tissue, in which endothelium is present, and endothelium-denuded (-E) tissue. LP-805-induced hyperpolarization was inhibited by glibenclamide. In -E tissues in both regions, acetylcholine (ACh, greater than 0.1 microM) depolarized the membrane, and LP-805 inhibited the depolarization. However, in +E tissues, ACh (greater than 0.1 microM) transiently hyperpolarized the membrane that was not modified by glibenclamide (10 microM), charybdotoxin (100 nM), and NG-nitro-L-arginine (L-NNA, 100 microM). In -E tissues of both regions, LP-805 consistently inhibited the 10 microM ACh-induced contraction (IC50, 2.8 microM), and 10 microM glibenclamide shifted this concentration-response curve to the right (IC50, 20 microM). In +E tissues, LP-805 more potently inhibited the ACh-induced contraction (IC50, 0.3 microM), and this inhibition was prevented by L-NNA (100 microM) but not by indomethacin or glibenclamide (10 microM). In -E and +E tissues of both regions, LP-805 repolarized the high K(+)-induced depolarization (less than 20 mM) and relaxed the tissues precontracted by high K+ (less than 30 mM); these electrical and mechanical effects of LP-805 were prevented by glibenclamide (10 microM) in +E tissues. In +E tissues, the K(+)-induced contraction (less than 30 mM) was more strongly inhibited than in -E tissues, but after treatment with L-NNA, LP-805 relaxed -E and +E tissues precontracted to the same extent in the presence of high K+. LP-805 (10 microM) did not inhibit the Ca(2+)-induced contraction in skinned muscle tissues but did slightly inhibit the ACh-induced contraction in Ca(2+)-free solution containing 2 mM EGTA. Thus, LP-805 has a potent releasing action on endothelium-derived relaxing factor and also the potential to open the glibenclamide-sensitive K+ channel. These events would account for the dilation of the rabbit coronary artery exposed to LP-805.     

Regulation of airway smooth muscle cell contractility by Ca2+ signaling and sensitivity

Airway smooth muscle cell contraction is regulated by changes in intracellular Ca2+ concentration ([Ca2+]i) and the responsiveness of the airway smooth muscle cell to this Ca2+. The mechanism controlling [Ca2+]i primarily involves agonist-induced release of Ca2+ from internal stores to generate Ca2+ oscillations. The extent of contraction correlates with the persistence and frequency of these Ca2+ oscillations. The maintenance of the Ca2+ oscillations requires Ca2+ influx, but membrane depolarization appears to have a minor role in initiating or sustaining contraction. Contraction also requires agonist-induced Ca2+ sensitization, which is mediated mainly by decreases in myosin light-chain phosphatase activity. Although it is not clear if airway hyperresponsiveness associated with asthma results from the specific modulation of these Ca2+-based regulatory mechanisms, bronchodilators relax airways by both attenuating the Ca2+ oscillations and by decreasing the Ca2+ sensitivity.     

Role of voltage-dependent and Ca(2+)-activated K(+) channels on the regulation of isometric force in porcine coronary artery

We investigated the role of K(+) channels in the regulation of vascular tone in de-endothelialized porcine coronary artery. Isometric force and intracellular Ca(2+) ([Ca(2+)](i)) under resting conditions were increased by treatment with 4-aminopyridine (4-AP, 1 mM), an inhibitor of voltage-dependent K(+) (K(v)) channels, but not by tetraethylammonium chloride (TEA, 1 mM) or charybdotoxin (100 nM), both inhibitors of Ca(2+)-activated K(+) (K(Ca)) channels, or glibenclamide (10 microM), an inhibitor of ATP-sensitive K(+) channels. Under stimulated conditions with 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619), 4-AP as well as TEA or charybdotoxin increased isometric force and [Ca(2+)](i), but not glibenclamide. 4-AP was the most potent in terms of depolarization of membrane potential compared with TEA or glibenclamide in the presence or absence of EGTA. In the presence of U46619, a high concentration of 4-AP (10 mM) caused a further contraction with oscillations. The force oscillations induced by 4-AP were inhibited by diltiazem (10 microM), an inhibitor of voltage-dependent Ca(2+) channels, or TEA (1 mM), but not by glibenclamide (10 microM). These force oscillations may be associated with the periodic activation of K(Ca) channels. These findings suggested that 4-AP-sensitive K(v) channels play an important role in the control of vascular tone in both resting and stimulated conditions. Moreover, under stimulated conditions, K(Ca) channels also have an important role in the regulation of vascular tone. Dysfunction of these channels induces abnormal vasoconstriction and may be implicated in vascular diseases such as hypertension and vasospasm.     

Electromechanical effects of leukotriene D4 on ferret tracheal muscle and its muscarinic responsiveness

We investigated the possible electrophysiological processes by which leukotriene D4 (LTD4) affects airway smooth muscle and its responsiveness to acetylcholine (ACh). For study in vitro, preparations of ferret tracheal muscle (dissected free of overlying mucosal and submucosal layers) were used. These preparations were arranged so that force transducers and glass intracellular microelectrodes (having tip resistances of 35-60 megohm) could be used to measure isometric force generation and cell membrane potential (Em) simultaneously from muscle stimulated by LTD4. At rest, the muscle was electrically and mechanically quiescent and had an Em of -59 +/- 0.2 mV (mean +/- SEM). We found that ferret tracheal muscle cells were relatively sensitive to LTD4, and that both the resulting depolarization (beginning at 10(-10) M LTD4) and force generation (produced by higher concentrations) progressed in a concentration-dependent manner. Depolarization by 10(-9) M LTD4 elicited electrical oscillations. These oscillations were accompanied by phasic contractile activity at 5 x 10(-9) M LTD4. Verapamil abolished these oscillations and diminished force substantially. We also found that ACh depolarized and contracted the muscle in a concentration-dependent manner. It caused electrical oscillations at greater than or equal to 10(-6) M. Diltiazem abolished these oscillations and markedly diminished force generation without affecting Em. Preexposure of airway muscle preparations for 20 min to a concentration (10(-10) M) of LTD4 that, by itself, did not produce significant force, substantially augmented the voltage-tension relationship of the muscle upon ACh stimulation. We conclude that there is an electrical basis for the slow, prolonged force generation of airway muscle caused by LTD4, and that LTD4 potentiates the electromechanical responsiveness of the airway muscle to muscarinic stimulation.     

Effect of pinaverium bromide on stress-induced colonic smooth muscle contractility disorder in rats

AIM: To investigate the effect of pinaverium bromide, a L-type calcium channel blocker with selectivity for the gastrointestinal tract on contractile activity of colonic circular smooth muscle in normal or cold-restraint stressed rats and its possible mechanism. METHODS: Cold-restraint stress was conducted on rats to increase fecal pellets output. Each isolated colonic circular muscle strip was suspended in a tissue chamber containing warm oxygenated Tyrode-Ringer solution. The contractile response to ACh or KCl was measured isometrically on ink-writing recorder. Incubated muscle in different concentrations of pinaverium and the effects of pinaverium were investigated on ACh or KCl-induced contraction. Colon smooth muscle cells were cultured from rats and (Ca(2+))(i) was measured in cell suspension using the Ca(2+) fluorescent dye fura-2/AM. RESULTS: During stress, rats fecal pellet output increased 61 % (P<0.01). Stimulated with ACh or KCl, the muscle contractility was higher in stress than that in control. Pinaverium inhibited the increment of (Ca(2+))(i) and the muscle contraction in response to ACh or KCl in a dose dependent manner. A significant inhibition of pinaverium to ACh or KCl induced (Ca(2+))(i) increment was observed at 10(-6) mol/L. The IC(50) values for inhibition of ACh induced contraction for the stress and control group were 1.66X10(-6) mol/L and 0.91X10(-6) mol/L, respectively. The IC(50) values for inhibition of KCl induced contraction for the stress and control group were 8.13X10(-7) mol/L and 3.80X10(-7) mol/L, respectively. CONCLUSION: Increase in (Ca(2+))(i) of smooth muscle cells is directly related to the generation of contraction force in colon. L-type Ca(2+) channels represent the main route of Ca(2+) entry. Pinaverium inhibits the calcium influx through L-type channels; decreases the contractile response to many kinds of agonists and regulates the stress-induced colon hypermotility.     

Electromechanical effects of tetraethylammonium and K+ on histamine-induced contraction in pig isolated tracheal smooth muscle

The effect of tetraethylammonium (TEA) and K⁺ on contractions to histamine and acetylcholine have been compared in the pig isolated trachea using organ bath and sucrose-gap techniques. Histamine elicited weak contractions, compared with acetylcholine; however, these contractions were markedly potentiated by pretreatment with TEA (10 mM) or by raising the external KCl concentration to 30–50 mM. Neither TEA nor K⁺ increased the sustained depolarization evoked by histamine (or acetylcholine) although oscillatory depolarizations were often observed in the presence of TEA. Verapamil and a zero Ca²⁺ Krebs solution reduced contractions to histamine and reduced or abolished the effect of TEA and K⁺ on histamine-induced contractions. The results unmask different mechanisms of contraction for histamine and acetylcholine. Histamine-induced tone appears to be linked with mechanisms sensitive to TEA and high K⁺, possibly involving increased translocation of Ca²⁺ across the plasma membrane.     

Mechanisms of flunarizine-induced vasodilation in the rabbit mesenteric artery

The vasodilating effects of flunarizine on smooth muscle strips of rabbit mesenteric artery have been investigated and compared with those of nifedipine. Flunarizine (30-300 nM) dose-dependently inhibited Ca2+-induced contractions in Ca2+-free solution containing 100 mM K+. Double reciprocal analysis showed that this inhibition was either competitive at low concentrations (30-100 nM; nifedipine-like) or noncompetitive at high concentrations (0.3-1 microM). The latter seemed to be partly related to an inhibition of contractile proteins as estimated from Ca2+-induced contractions in saponin-treated chemically skinned muscle strips. In contrast to the actions of nifedipine, flunarizine inhibited norepinephrine (NE)-induced contractions more than those induced by high K+, and at 0.3 microM, this agent totally blocked NE-induced contraction. Flunarizine also inhibited NE-induced contraction in Ca2+-free solution containing 2 mM EGTA. In Ca2+-free solution, NE rapidly hydrolyzed phosphatidylinositol 4,5-bisphosphate (PI-P2) and produced phosphatidic acid (PA). Flunarizine (30 and 300 nM), but not nifedipine (100 nM), inhibited NE-induced hydrolysis of PI-P2 and production of PA. However, flunarizine (100 nM) did not modify the contraction induced by 10 microM inositol 1,4,5-trisphosphate in chemically skinned muscle strips. It is concluded that flunarizine inhibits both voltage-dependent (nifedipine-like) and receptor-operated Ca2+ influx induced by NE and also inhibits NE-induced Ca2+ release from intracellular stores due to inhibition of the hydrolysis of PI-P2.     

Phospholipase A2 augments contraction and intracellular calcium mobilization through thromboxane A2 in bovine tracheal smooth muscle.

Phospholipase A2 (PLA2) induces hyper-sensitivity to muscarinic agonists in airway smooth muscle in vitro. The precise mechanism of this is unknown, but might involve altered calcium homeostasis. In order to elucidate the effects of PLA2, on bovine tracheal smooth muscle contraction, isometric tension and intracellular calcium concentration ([Ca2+]i) were simultaneously measured in fura 2-loaded muscle strips. A high concentration of PLA2 (0.5 microg x mL(-1)) caused the muscle strips to contract, and this contractile response was significantly attenuated by pretreatment with indomethacin (IND; 10 microM), but not by nordihydroguaiaretic acid (NDGA; 10 microM). A low concentration of PLA2 (0.02 microg x mL(-1)) did not directly contract muscle strips. However a low concentration PLA2 significantly enhanced the threshold of the contractile response and that of the [Ca2+]i response to acetylcholine (ACh), but not that of the response to a high K+ concentration. These augmented responses to ACh returned to control levels after pretreatment with IND, a thromboxane (TX) synthetase inhibitor (OKY-046; 10 microM) or a TXA2 receptor antagonist (ONO-3708; 10 microM), but not after NDGA pretreatment. These results suggest that a low concentration of phospholipase A2 enhances smooth muscle responsiveness to acetylcholine by agonist-mediated Ca2+ mobilization facilitated by thromboxane A2. It is concluded that phospholipase A2 plays an important role in bronchial hypersensitivity involving thromboxane A2. It remains to be examined whether similar abnormalities in calcium homeostasis and muscarinic receptor function or coupling are involved in the pathogenesis of asthma.     

Azelastine's inhibition of histamine and tryptase release from human umbilical cord blood-derived cultured mast cells as well as rat skin mast cell-induced vascular permeability: comparison with olopatadine.

Mast cells are involved in early and late-phase reactions by releasing vasoactive molecules, proteases, and cytokines. Azelastine and olopatadine are histamine 1 receptor (H-1R) antagonists with antiallergic effects present in the ophthalmic solutions Optivar and Patanol, respectively. Because it is difficult to obtain animal or human conjunctival tissue, we first investigated the effect of these compounds on histamine and tryptase release from cultured human mast cells (CHMCs) grown out of human umbilical cord blood-derived CD34+ cells. Sensitized CHMCs were pretreated with various concentrations of azelastine or olopatadine for 5 minutes. Then, CHMCs were challenged with anti-immunoglobulin E (IgE) and the released mediators were quantitated. The greatest inhibition of mediator release was seen when CHMCs were pretreated with 24 microM of azelastine or 133 microM of olopatadine (2% dilution of azelastine or 5% olopatadine original ophthalmic solutions, respectively). We then studied the drug concentrations that gave optimal results on skin vasodilation induced by the mast cell secretagogue compound 48/80. An intradermal injection of 48/80 in rats, to which Evan's blue had been administered via the tail vein, induced substantial dye extravasation. Pretreatment of the injection site for 5 minutes with either 24 microM of azelastine or 133 microM of olopatadine completely prevented extravasation; this effect was quantitated also by fluorometric assessment of Evan's blue extracted in formamide. Evaluation of skin mast cells from injected sites showed that mast cell degranulation was inhibited greatly. These results indicate that on an equimolar basis, azelastine was a more potent inhibitor than olopatadine of both CHMC and rat skin mast cells activation.     

Effect of histamine on electrical properties of airway epithelium and its antagonism by azelastine]

To elucidate the mechanism of the action of histamine on bioelectric properties of the airway epithelium and the fact that the anti-allergic agent azelastine is possibly antagonistic to it, we studied canine cultured tracheal epithelium under short-circuit conditions in vitro. Addition of histamine to the submucosal but not mucosal bath of Ussing chamber increased short-circuit current (Isc) in a dose-dependent fashion, the maximal increase and the EC50 value being 5.7 +/- 0.9 microA/cm2 (mean +/- SE, p less than 0.001) and 3 x 10(-6) M, respectively. Pretreatment of tissues with pyrilamine abolished the histamine-induced increase in Isc (p less than 0.001), whereas cimetidine had no effect. In addition, the histamine action on Isc was effectively depressed by the Cl channel blocker diphenylamine-2-carboxylate, Cl-free medium, and indomethacin. The release of PGE2 and PGF2 alpha was significantly enhanced by the submucosal application of histamine. Pretreatment of tissues with azelastine dose-dependently attenuated the increase in Isc induced by 10(-4) M histamine. These results indicate that histamine increases the airway epithelial Isc via stimulation of prostaglandin synthesis and subsequent movement of Cl toward the lumen, and that azelastine may be useful in preventing histamine-induced water secretion in the airway.     

Effects of procaine on pharmaco-mechanical coupling mechanisms activated by acetylcholine in smooth muscle cells of porcine coronary artery

The action of procaine on pharmaco-mechanical coupling activated by application of acetylcholine (ACh) was investigated using collagenase-treated dispersed intact and skinned smooth muscle cells and intact muscle tissues of the porcine coronary artery. ACh reduced stored 45Ca2+, and this action was prevented by procaine in intact dispersed cells. The maximum reduction in the level of stored 45Ca induced by caffeine (25 mM) or inositol 1,4,5-trisphosphate (InsP3; 3 microM) was also prevented by procaine in the skinned muscle cells in the presence or absence of ATP. However, inhibitions of the latter required higher concentrations of procaine than the former. Release by 10 microM ACh of Ca2+ from its store site in the presence or absence of extracellular Ca2+ was also inhibited by procaine and was detected using the quin2 fluorescence method. In these smooth muscle tissues, ACh (above 10 nM) reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and dose dependently increased the amount of phosphatidic acid. Procaine inhibited the hydrolysis of PI-P2 activated by ACh, thus reducing the amount of InsP3 and the release of Ca2+ from the store site. It is concluded that procaine has multiple actions on the porcine coronary artery, and one of the actions related with pharmacomechanical coupling appears through inhibition of hydrolysis of PI-P2 induced by ACh.     

Relation between membrane potential and contractile force in smooth muscle of the rat tail artery during stimulation by norepinephrine, 5-hydroxytryptamine, and potassium

The relation between smooth muscle membrane potential and contractile force was investigated in the rat tail artery to assess the importance of smooth muscle depolarization in the control of smooth muscle tone. Smooth muscle membrane potential and contractile force were measured simultaneously in isolated pieces of rat tail artery exposed to a range of concentrations of norepinephrine, 5-hydroxy-tryptamine, or raised external potassium. Potassium caused depolarization and contraction when the membrane was depolarized beyond -40 mV. Maximum contraction occurred at -19 mV, and further depolarization gave no increase in contraction. Both norepinephrine and 5-hydroxytryptamine caused contraction and depolarization, but the relation between depolarization and contraction was not the same as when potassium was used. There was significant contraction when the membrane potential was more negative than -50 mV, and the membrane potential was around -30 mV during maximal contractions. Although they acted on pharmacologically different membrane receptors, the relation between membrane potential and contraction was the same for norepinephrine and 5-hydroxytryptamine. Prazosin reduced the responses to norepinephrine but did not change the relation between membrane potential and contractile force. These results indicated that norepinephrine and 5-hydroxytryptamine binding to their respective receptors might activate the same sets of intracellular processes that subsequently caused both depolarization and contraction.     

Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx

Mouse pancreatic islets were used to investigate the mechanisms and functional significance of the B cell membrane depolarization by acetylcholine (ACh). At low glucose (3mM), ACh (20 microM) increased 22Na+ influx, and slightly depolarized the B cell membrane but did not induce electrical activity or stimulate 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and barely affected basal insulin release. At a stimulatory concentration of glucose (10 mM), ACh stimulated 22Na+ influx, depolarized the B cell membrane, increased glucose-induced electrical activity, and stimulated 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and strongly potentiated insulin release. Omission of extracellular Ca2+ did not impair ACh stimulation of 22Na+ influx or 86Rb+ efflux, slightly modified the acceleration of 45Ca2+ efflux, and almost completely suppressed the increase in insulin release. Na+ omission (with N-methyl-D-glucamine as substitute) prevented the B cell membrane depolarization and the stimulation of 45Ca2+ influx, largely inhibited the acceleration of 86Rb+ efflux and insulin release, and suppressed the late phase of 45Ca2+ efflux otherwise produced by ACh. On the other hand, ACh stimulation of 3H efflux from islets prelabeled with myo-[2-3H]inositol was not affected by Na+ omission. All effects of ACh were blocked by atropine and unaffected by nicotinic antagonists. It is concluded that activation of muscarinic receptors depolarized the B cell membrane by increasing its permeability to Na+. When the membrane is already depolarized by glucose, this further depolarization augments Ca2+ influx and, hence, potentiates insulin release.     

Electromechanical effects of leukotriene D4 on ferret tracheal muscle and its muscarinic responsiveness

We investigated the possible electrophysiological processes by which leukotriene D₄ (LTD₄) affects airway smooth muscle and its responsiveness to acetylcholine (ACh). For study in vitro, preparations of ferret tracheal muscle (dissected free of overlying mucosal and submucosal layers) were used. These preparations were arranged so that force transducers and glass intracellular microelectrodes (having tip resistances of 35–60 megohm) could be used to measure isometric force generation and cell membrane potential (Em) simultaneously from muscle stimulated by LTD₄. At rest, the muscle was electrically and mechanically quiescent and had an Em of −59±0.2 mV (mean±SEM). We found that ferret tracheal muscle cells were relatively sensitive to LTD₄, and that both the resulting depolarization (beginning at 10⁻¹⁰ M LTD₄) and force generation (produced by higher concentrations) progressed in a concentration-dependent manner. Depolarization by 10⁻⁹ M LTD₄ elicited electrical oscillations. These oscillations were accompanied by phasic contractile activity at 5 × 10⁻⁹ M LTD₄. Verapamil abolished these oscillations and diminished force substantially. We also found that ACh depolarized and contracted the muscle in a concentration-dependent manner. It caused electrical oscillations at ≥ 10⁻⁶ M. Diltiazem abolished these oscillations and markedly diminished force generation without affecting Em. Preexposure of airway muscle preparations for 20 min to a concentration (10⁻¹⁰ M) of LTD₄ that, by itself, did not produce significant force, substantially augmented the voltage-tension relationship of the muscle upon ACh stimulation. We conclude that there is an electrical basis for the slow, prolonged force generation of airway muscle caused by LTD₄, and that LTD₄ potentiates the electromechanical responsiveness of the airway muscle to muscarinic stimulation.     

Caffeine rapid cooling contractures and negative force staircase in rat papillary muscle.

In rat papillary muscle, rapid cooling causes membrane depolarization which initiates action potentials that lead to a contraction. This rapid cooling contraction (RCC) can be blocked by TTX, Mn2+, Ni2+ or high K+ superfusion. In the presence of caffeine (0.5-1 mM), the rapid cooling contracture (caffeine-RCC) has an amplitude similar to that of a twitch elicited by field stimulation at 37 degrees C, but is not inhibited by these agents. As the caffeine-RCC appears to be independent of membrane depolarization and Ca influx but can be inhibited by increasing the bathing caffeine concentration to 20 mM, we consider that the amplitude of this contracture gives a good indication of the calcium content of the sarcoplasmic reticulum (SR). In Tyrode containing 1.8 mM Ca an increased stimulus frequency leads to a negative force staircase which is paralleled by a similar decrease in the amplitude of the caffeine-RCC. These effects are lost if the bathing Ca is reduced (0.18-0.45 mM) in a way which can be reversed by isoproterenol (100 nM). In verapamil (2 microM), however whilst the twitch responses may show a steeper dependence upon stimulus frequency, the negative frequency dependence of the caffeine-RCC is also lost. Low external Na+ also inhibits the frequency dependent reduction of the caffeine-RCC. The results suggest that if the amplitude of the caffeine-RCC is a good indication of the SR calcium content, then this Ca store is related reciprocally to membrane Ca current where activation of the Ca channels leads to a depletion of the store whereas inhibition of membrane Ca channels leads to a filling of the Ca store. We propose that on stimulation the size of the Ca influx determines the fraction of Ca released from the SR. This released Ca may be partially extruded from the cell by way of the Na/Ca exchange which acts in competition with the re-uptake mechanism of the SR to control SR Ca content.     

Current fluctuations and oscillations in smooth muscle cells from hog carotid artery. Role of the sarcoplasmic reticulum

Electrical activity of enzymatically isolated, smooth muscle cells from hog carotid arteries was recorded under current clamp and voltage clamp. Under the experimental conditions, membrane potential usually was not stable, and spontaneous hyperpolarizing transients of approximately 100-msec duration were recorded. The amplitude of the transients was markedly voltage dependent and ranged from about 20 mV at a membrane potential of 0 mV to undetectable at membrane potentials negative to -60 mV. Under voltage clamp, transient outward currents displayed a similar voltage dependency. These fluctuations reflect a K+ current; they were abolished by 10 mM tetraethylammonium chloride, a K+ channel blocker, and the current fluctuations reversed direction in high extracellular K+ concentration. Modulators of intracellular Ca2+ concentration also affected electrical activity. Lowering intracellular Ca2+ concentration by addition of 10 mM EGTA to the pipette solution or suppressing sarcoplasmic reticulum function by superfusion with caffeine (10 mM), ryanodine (1 microM), or histamine (3-10 microM) blocked the rapid voltage and current spikes. However, caffeine and histamine induced a much slower hump of outward current before blocking the rapid spikes. This slower transient outward current could be elicited only once after external Ca2+ was removed and is consistent with an activation of K+ channels by Ca2+ released from internal stores. In contrast, removal of external Ca2+ alone failed to abolish the rapid spikes. These results suggest that 1) a Ca2+-dependent K+ conductance can markedly affect the electrical behavior of arterial smooth muscle cells and 2) internal Ca2+ stores, probably the sarcoplasmic reticulum, can support rapid and frequent releases of Ca2+. Exposure to a low concentration of histamine (3 microM) caused synchronization of the irregular, rapid fluctuations giving rise to slow, periodic oscillations of Ca2+-activated K+ conductance with a frequency of 0.1-0.3 Hz. These regular oscillations are reminiscent of periodic Ca2+-induced Ca2+ release, were inhibited by 10 mM caffeine, and point to a modulation of sarcoplasmic reticulum Ca2+ release by histamine.     

Role of Ca2+- and swelling-activated Cl- channels in alpha1-adrenoceptor-mediated tone in pressurized rabbit mesenteric arterioles

BACKGROUND: Ca2+-activated (I(Cl(Ca))) and swelling-induced (I(Cl(swell))) Cl- channels have, respectively, been postulated to participate in the membrane depolarization and contraction mediated by activation of alpha1-adrenoceptors and vascular wall distension during pressurization. Their respective function in generating active force in pressurized arterioles during alpha1-adrenoceptor stimulation remains unsettled. OBJECTIVES: Experimental protocols were designed to: (1) assess the relative contribution of I(Cl(Ca)) to the pressure-dependence of lumen diameter of mesenteric arterioles at different states of activation of the alpha1-adrenoceptor, and (2) investigate the potential role of I(Cl(swell)) in spontaneous and agonist-mediated myogenic reactivity. METHODS: Segments of endothelium-denuded rabbit mesenteric arterioles with a lumen diameter of approximately 70 microm were cannulated at both ends and studied under isobaric conditions at 36 degrees C. Steady-state lumen diameter at each pressure step investigated (0-100 mmHg, in 20-mmHg increments) was measured by a video-microscopy edge-detection technique. RESULTS: Under control conditions, 23% of the arterioles developed nifedipine-sensitive spontaneous myogenic tone. In the presence of 1 mM tetraethylammonium chloride (TEA) to inhibit Ca2+-dependent K+ channels, the alpha1-agonist phenylephrine (PE) contracted the vessels in a concentration-dependent manner (0.1-10 microM) and potentiated myogenic reactivity. The contraction mediated by 1 microM PE/TEA was abolished by 1 microM nifedipine, indicating that Ca2+ entry through voltage-gated Ca2+ channels was a necessary step in the cascade leading to contraction. Niflumic acid (NfA, 100 microM), a relatively selective inhibitor of I(Cl(Ca)), had no effect on myogenic tone but reversed the PE-induced contraction, varying with the concentration of PE and transmural pressure. For PE concentrations between 0.1 and 1 microM, but not for 10 microM PE, the relaxing efficacy of NfA decreased as applied pressure was raised from 0 to 100 mmHg. At all pressure steps, the NfA-induced relaxation was inversely related to the concentration of PE. DIDS (200 microM), another Cl- channel blocker, inhibited spontaneous myogenic tone, and partially suppressed a component of contraction at elevated transmural pressures in arterioles incubated in 1 microM PE/1 mM TEA/100 microM NfA. CONCLUSIONS: Our data indicate that under low to moderate stimulation of the alpha1-adrenoceptor signaling pathway, I(Cl(Ca)) channels play an important role in the sustained contraction produced. Their declining contribution to contraction with increasing transmural pressure may be explained, at least in part, by a progressive enhancement of stretch-induced ionic conductances, possibly volume-sensitive Cl- channels.