Effects of azelastine on vagal neuroeffector transmission in canine and human airway smooth muscle

Azelastine is a newly developed antiallergic drug that is reported to antagonize histamine and leukotrienes in addition to its inhibitory action on release of chemical mediators. In the present study, the effects of azelastine on neuroeffector transmission in the airway smooth muscles with double sucrose gap and isometric tension-recording methods were evaluated. Azelastine (10(-8) to 10(-6) mol/L) markedly decreased the contractile response of human bronchial and dog tracheal muscle strips to electrical field stimulation (10 pulses at 20 V, 20 Hz, 800 microseconds) in a dose-dependent manner. In parallel with actions on twitch contractions, azelastine suppressed the amplitude of excitatory junction potentials of dog trachea without changing the resting membrane potential and input resistance of smooth muscle cells. However, azelastine did not alter acetylcholine sensitivity of the smooth muscle cells. These results indicate that azelastine possesses an inhibitory action on the release of acetylcholine by vagal nerve terminals. This inhibitory effect of azelastine may contribute to the treatment of asthma in addition to its antiallergic actions.     

Adenosine relaxation of isolated vascular smooth muscle.

Adenosine relaxed hog carotid media strips contracted with norepinephrine (NE) and potassium (K+). Adenosine (3 X 10(-6)M) was more effective in relaxing the NE contractures than those produced by K+. In both cases, adenosine's efficacy decreased with increasing concentrations of the stimulating agent. A high adenosine concentration (1 X 10(-3)M) was necessary to elicit relaxation of completely depolarized (124 mM K+) media strips and equimolar concentrations of aminophylline caused greater relaxation than did adenosine. Adenosine inhibited the Ca2+ dose-response curves of strips stimulated with 20 mM and 30 mM K+ and its effect was dependent on the Ca2+ concentration. Neither 1 X 10(-6)M nor 1 X 10(-4)M adenosine produced any change in the cAMP content of vascular strips. Only at high concentrations did adenosine increase the cAMP content of vascular strips, but the increase was signficantly more than that observed with the same dose of aminophylline. The present results are consistent with the possibility that adenosine relaxes vascular smooth muscle by directly altering Ca2+ permeability and/or membrane potential; they do not support a role for cAMP in the adenosine-induced relaxation of vascular smooth muscle.     

Ionic currents in the uterine smooth muscle.

1. Short segments of isolated longitudinal myometrium from the pregnant rate uterus have been studied in a double sucrose-gap voltage-clamp arrangement. The clamped segment averaged 65 mum times 240 mum times 100 mum, has an average total capacitance of 0-14 muF, and may contain 50-200 individual myometrial cells. 2. A significant resistance exists in series with the membrane, and limits theprecision of the quantitative information. However, it is argued that some qualitative and some comparative information is useful. 3. In Krebs-bicarbonate solution, depolarizing steps produced initial transient inward currents followed by delayed outward currents. 4. When [Na+]o was reduced by 50%, the equilibrium potential Ea shifted by an average of -17-6 mV, the maximum inward current was reduced to 0-5, the time to peak of the early current was delayed by 1-1 msec, and the maximum chord conductances for the early(Ga) and late (GK) currents remained unchanged as compared with those in normal [Na+]o. 5. When [Ca2+] was reduced to 25% of normal, Ea shifted by an average of -20-3 mV, the maximum inward current was reduced to 0-5, the time to peak was delayed 3-1 msec, and Ga was significantly reduced, while GK was unaffected. 6. The early current, and its tail when repolarization was imposed, reversed direction from inward to outward when [Na+]o was reduced from 143 mM to zero, with [Ca2+]o remaining constant at 1-9 mM. 7. From the observations in 4, 5 and 6, it was concluded that Na+ is the main charge carrier for the early current, and that Ca2+ is important in regulating Ga. 8. The late current is outwards when [K+]o equals 5-9 mM, but inwards in some voltage range when [K+]o was elevated to 120 or 148 mM. K+ is the main charge carrier for the late current. 9. The equilibrium potential for the late current, EK, is about 15 mV more negative than the natural resting potential. 10. Prolonged holding of the preparations at voltages that differ significantly from the natural resting potential tends to shift EK in a way consistent with passive changes in [K+]i by the holding current. 11. The steady-state inactivation of the early current, h, is unusual. Inward current is macimum around the resting potential, and declines with both hyperpolarizing and depolarizing changes. Half-inactivation occurred with about 9 mV depolarization and 15 mV hyperpolarization. 12. The instantaneous current-voltage relations of both early and late currents are linear. The chord conductances Ga and GKare similar in form to those in other tissues.     

Ionic fluxes in rat uterine smooth muscle.

1. The K+, Na+ and Cl-fluxes from the oestrogen-stimulated rat uterine smooth muscle were measured using radioactive tracers. 2. The cellular compartment contained a concentration of K+ of 173-6 mM which exchanged at a rate of 5-9 x 10(-12) mol.cm-2.sec-1. 3. Cl- exchanged at a rate of 6-9 x 10(-12) mol.cm-2.sec-1 from a cellular compartment having a concentration of 39-3 mM. 4. The methods used for the evaluation of Na+ movements over-estimate both influx and efflux values. If an average value of flux of 9-2 x 10(-12) mol.cm-2.sec-1 is considered we obtain PNa+/PK+ ratios of 2-4 (-42 mV) or 1-3 (-57-6 mV), which are too high and do not correspond to electrophysiological evidence. 5. The relative permeabilities PCl-/PK+ in the case of a membrane potential of -42-0 mV could be 0-8, or 1-3 in the case of a membrane potential of -57-6 mV. 6. Both conductances GK+ and GCl- seem to play an important role in determining membrane conductance.     

Spontaneous activity and its cholinergic modulation in circular smooth muscle isolated from guinea-pig stomach antrum

Circular smooth muscle isolated from the guinea-pig gastric antrum generated periodic slow potentials in the presence of nifedipine and nitroarginine to prevent the activity of voltage-gated L-type Ca-channels and endogenous production of NO respectively. Chelerythrine, an inhibitor of protein kinase C (PKC), in the concentration range 10^–7–3×10^–7 M reduced the frequency but not the amplitude of spontaneous slow potentials without altering the resting membrane potential. 2-Aminoethoxydiphenyl borate (2-APB, 3×10^–6 M), an inhibitor at inositol-1,4,5-trisphosphate (IP₃) receptors, depolarized the membrane, increased the frequency and reduced the amplitude of the slow potentials; the latter actions were independent of depolarization. Two different phorbol esters, phorbol 12,13-dibutyrate and phorbol-12-myristate-13-acetate, increased the frequency of slow potentials, without altering the amplitude or changing the resting membrane potential; the effects of phorbol esters were antagonized by chelerythrine. Stimulation of muscarinic receptors with acetylcholine (ACh), in concentrations below those causing membrane depolarization (3×10^–8–10^–7 M), increased the amplitude and frequency of slow potentials. Chelerythrine inhibited the ACh-induced increase in the frequency of slow potentials but did not prevent the increase in their amplitude. 2-APB inhibited the ACh-induced increase in the amplitude of slow potentials but did not prevent the increase in their frequency. These results suggest that the frequency of spontaneous slow potentials is regulated by PKC and their amplitude by IP₃ production. ACh increases both the amplitude and frequency of slow potentials; the former is related to the activation of PKC, while the latter is related to activation of IP₃-receptors.     

Adenosine induces prolonged anti-beta-adrenergic effects in guinea-pig papillary muscle

A sustained anti-beta-adrenergic effect of adenosine has been reported. This study was initiated to investigate this topic and especially elucidate the role of protein kinase C (PKC). Contractile force amplitude and action potential duration at 90% repolarization (APD90) were measured in guinea-pig papillary muscles before and after 5 min challenge with 5 nm isoproterenol. Protocols contained 30 min exposure to the test agents adenosine 33 microm (ado), adenosine + PKC-inhibitor bisindolylmaleimide 20 nM (ado + BIM), PKC-activator 1,2-dioctanoyl-sn-glycerol 10 microm (DOG) and alpha-agonist phenylephrine 5 microm (phe). Isoproterenol was given at the end of test exposure and after 15 min washout. Results are mean +/- SEM of percentage-change, P < or = 0.05 considered significant and labelled *. The first isoproterenol challenge significantly increased contractile force (27 +/- 7%*) in the control group. Responses in the test groups were 2 +/- 4 (ado), 1 +/- 5 (ado + BIM), 14 +/- 4* (DOG), 0 +/- 2% (phe). After washout of adenosine, DOG and phenylephrine, isoproterenol induced 3 +/- 8 (ado), 23 +/- 5* (ado + BIM), 13 +/- 5* (DOG), 15 +/- 7% (phe) increase in test groups compared with 22 +/- 5%* increase in contractile force in the control group. After 45 min washout of adenosine the inotropic response was still significantly reduced compared with control (29 +/- 4 vs. 79 +/- 8%*). Isoproterenol stimulation shortened APD90 in controls at both time points (5 +/- 1%* and 4 +/- 1%*), with no significant shortening in test groups. Adenosine induces sustained anti-beta-adrenergic effects on contractile force as well as APD90. A role for PKC in signal transduction is supported with respect to contractile force.     

Modulation of neuroeffector transmission in guinea-pig pulmonary artery and vas deferens by exogenous nitric oxide

Cederqvist , B. & Gustafsson , L. E. 1994. Modulation of neuroeffector transmission in guinea-pig pulmonary artery and vas deferens by exogenous nitric oxide. Acta Physiol Scand 150, 75–81. Received 11 March 1993, accepted 30 July 1993. ISSN 0001–6772. Department of Physiology and Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden. Blockade of nitric oxide (NO) synthesis enhances contractile responses to transmural nerve stimulation in guinea-pig pulmonary artery, indicating neuromodulation by endogenous nitric oxide. In the present study, neuromodulatory effects of exogenous NO were examined in guinea-pig pulmonary artery and vas deferens. Application of NO as acid nitrite, to the guinea-pig pulmonary artery, inhibited the contractile response to transmural nerve stimulation as well as contractions to exogenous noradrenaline. The inhibition occurred in a dose-dependent manner. Acid nitrite did not affect stimulation-induced release of [³H]noradrenaline in guinea-pig pulmonary artery. In the guinea-pig vas deferens, contractile ‘twitch’ responses to brief (25 pulses) transmural nerve stimulation and stimulation-induced release of [³H]noradrenaline was unaffected by acid nitrite. However, ‘twitch’ responses to prolonged stimulation were inhibited by acid nitrite, and tonic ‘hump’ responses were either enhanced or unaffected by acid nitrite. In conclusion, exogenous nitric oxide exerted an inhibition of adrenergic neurotransmission post-junctionally in guinea-pig pulmonary artery. In guinea-pig vas deferens, exogenous nitric oxide affected adrenergic and/or non-adrenergic non-cholinergic neurotransmission in a complex fashion, however without alteration in noradrenaline release. The data support a role for NO in modulation of neuroeffector transmission, especially by modulation of effector sensitivity, whereas modulation of noradrenaline release seems an unlikely role for NO in these and similar peripheral tissues.     

Transmission from vasoconstrictor and vasodilator nerves to single smooth muscle cells of the guinea-pig uterine artery

1. Previous studies have shown that perivascular nerve stimulation of the uterine artery of the guinea-pig evokes an adrenergic constrictor response and a dilator response with two components. The first of these, only present during pregnancy, is cholinergic. The second is non-cholinergic and is present at all times. Intracellular recording from single smooth muscle cells in isolated arterial segments has now been used to investigate the transmission processes associated with these responses.2. The mean resting membrane potential of the muscle cells was 60.7 mV in arteries from both virgin animals (range 50-68 mV) and from animals in late pregnancy (range 48-76 mV).3. Low frequency perivascular stimulation evoked excitatory junction potentials (EJPs) which reached a maximum amplitude of about 5 mV, lasted about 900-1000 msec, and showed facilitation at frequencies of stimulation of 0.1 Hz or above and summation at frequencies of stimulation of 1.2 Hz or above.4. These EJPs were abolished by exposure of the tissue to bretylium (2 x 10(-6) g/ml.). It is therefore concluded that the EJPs were due to transmission from adrenergic nerves.5. Perivascular stimulation at frequencies above 10 Hz evoked a depolarizing response which was often surmounted by a small (5 mV) local spike potential. Such depolarizing responses were associated only with localized contractions of the arterial muscle.6. In the presence of low extracellular K(+) concentrations, perivascular stimulation at frequencies above 10 Hz gave rise to a depolarizing response topped by an action potential of up to 50 mV amplitude, and more generalized contraction of the tissue than was seen in normal K(+) solution.7. After blockade of the adrenergic vasoconstrictor fibres, no response to perivascular stimulation was observed normally. However, following moderate depolarization of the membrane with noradrenaline, stimulation evoked a hyperpolarization of up to 6 mV in amplitude.8. This response showed no discrete junction potentials, had a latency of up to 2000 msec and was only observed with stimulation at frequencies of 2 Hz or greater. The response was obtained in both pregnant and non-pregnant animals, and was unaffected by hyoscine, but was abolished by cinchocaine.9. No changes in membrane potential attributable to transmission from cholinergic dilator nerves could be revealed in arteries from pregnant animals. Furthermore, high concentrations of acetylcholine had no polarizing effect on the muscle cells. It is suggested that the cholinergic dilator nerves may not act via changes in membrane potential.     

Calcium and angiotensin tachyphylaxis in rat uterine smooth muscle.

Studies were carried out to investigate the relationship between extracellular Ca++ and the ability of a particular smooth muscle to develop tachyphylaxis to angiotensin II (AII). Stimulation of rat uterus by AII was found to be dependent on extracellular Ca++. Placing the tissue in 0 Ca++ completely blocked AII-induced contractions as did the presence of the "Ca++ antagonists" verapamil (10- minus 5M), SKF 525-A (10- MINUS 5M), tetracaine (10- minus 4M), Mn++ (8 times 10- minus 3M), or La-3+ (10- minus 3M). In addition, it is no longer possible to produce tachyphylaxis to AII in deplorazed rat uterus under conditions of pH and Ca++ concentration in which a normally polarized preparation would be unresponsive. Verapamil, on the other hand, was an even more effective antagonist of AII in depolarized preparations (ID50 of 10- minus 8M) than in normal tissues (ID50 of 2.0 times 10- minus 7M). Like the rat uterus, the smooth muscle of the guinea pig ileum also develops tachyphylaxis to AII, and the effect of this peptide was also blocked by 10- minus 5 M verapamil. Rabbit aorta, however, was found to be relatively resistant to both development of tachyphylaxis under conditions of low Ca++ and low pH and also to inhibition by even very high concentrations of verapamil (10- minus 4M). The results of these studies suggest that the Ca++ site involved in the tachyphylactic response to AII may be a physiologically important one in those smooth muscles in which movement of extracellular Ca++ contributes to the inward ion currents during excitation. Verapamil, however, appears to act at a common step in the excitation-contraction sequence in rat uterus. A working model of the interaction of AII with rat uterine smooth muscle is presented.     

Spike-free activation mechanism in smooth muscle of guinea-pig stomach

Electrical and mechanical activity of circular muscle strips of guinea-pigs stomach, taken from the distal corpus/proximal antrum region, were recorded. Spontaneous activity consisting of phasic contractions combined with bursts of spike potentials was suppressed by verapamil (5-10-6 - 2-10-5 mol/l). Under these conditions acetylcholine produced a spike-free tonic activation. Under normal conditions phasic contractions were superimposed on this tonic activation. The acetylcholine-induced activation, therefore, consists of two different components, one of which can be selectively blocked with verapamil. Both components disappear quickly in calcium-free solution. It can be concluded that two different calcium activation systems are responsible for the two components of activation. In comparative studies with taenia coli preparations a comparable spike-free tonic activation was not found.     

Electrical and mechanical properties and neuro-effector transmission in the smooth muscle layer of the guinea-pig ileocecal junction

Electrical and mechanical properties and neuro-effector transmission were studied in circular strips of smooth muscle taken from the ileocecal junction of guinea-pigs in relation to sphincter action, using the microelectrode, and tension recording methods. The membrane potential of the smooth muscle was low (–43 mV) compared with the membrane potential of circular muscle cells of the ileum or caecum (–58 mV or –62mV). Only small populations of the muscle cells (about 5%) generated spontaneous action potentials.Field stimulation of the tissue produced an initial slight relaxation followed by a contraction, and the mechanical responses were accompanied by membrane hyperpolarization (i. j. p.) followed by repolarization with rebound spikes. Treatment with atropine increased the amplitude of i.j. ps and decreased the amplitue of the rebound repolarization. Propranolol or phentolamine did not affect the amplitude of i. j. p., however, phentolamine slightly reduced the amplitude of the rebound repolarization.These results indicate that the ileocecal junction is predominantly controlled by non-adrenergic, non-cholinergic inhibitory nerve fibres and that the distribution of adrenergic and cholinergic excitatory nerve fibres is sparse.     

Pre- and postjunctional modulation of cholinergic neuroeffector transmission by adenine nucleotides. Experiments with agonist and antagonist

Adenosine, 2-chloroadenosine, AMP, AMPNH2, ADP, ATP, AMPPNP and beta, gamma meATP dose-dependently and reversibly inhibited contractile responses to nerve stimulation in longitudinal plexus-containing muscle preparations of guinea pig ileum. All the purines, except for 2-chloroadenosine, were virtually equipotent and elicited maximal inhibition within 2 min after application. Adenosine deaminase abolished inhibition by adenosine but did not block the effect of 2-chloroadenosine or the adenine nucleotides. During transmural nerve stimulation, the nucleotidase-resistant analogues alpha, beta meADP and AMPPNP significantly depressed acetylcholine release measured by gas chromatography-mass spectrometry. The inhibitory effect of all the purines was competitively, reversibly and at comparable pA2 values antagonized by 8-p-sulphophenyltheophylline. The results indicate that nucleotides per se can inhibit neurotransmission via a prejunctional receptor common to nucleosides and nucleotides. An excitatory effect by all the di- and triphosphate nucleotides was also observed. The excitation was unaltered by atropine, tetrodotoxin and 8-p-sulphophenyltheophylline, thus suggesting an action at postjunctional ADP- and ATP-like receptive sites.     

Ghrelin signalling in guinea-pig femoral artery smooth muscle cells

Aim: Our aim was to study the new signalling pathway of ghrelin in the guinea‐pig femoral artery using the outward I_K as a sensor. Methods: Whole‐cell patch‐clamp experiments were performed on single smooth muscle cells, freshly isolated from the guinea‐pig femoral artery. The contractile force of isometric preparations of the same artery was measured using a wire‐myograph. Results: In a Ca²⁺‐ and nicardipine‐containing external solution, 1 mmol L^?1 tetraethylammonium reduced the net I_K by 49 ± 7%. This effect was similar and not additive to the effect of the specific BK_Ca channel inhibitor iberiotoxin. Ghrelin (10^?7 mol L^?1) quickly and significantly reduced the amplitudes of tetraethylammonium‐ and iberiotoxin‐sensitive currents through BK_Ca channels. The application of 5 × 10^?6 mol L^?1 desacyl ghrelin did not affect the amplitude of the control I_K but it successfully prevented the ghrelin‐induced I_K decrease. The effect of ghrelin on I_K was insensitive to selective inhibitors of cAMP‐dependent protein kinase, soluble guanylyl cyclase, cGMP‐dependent protein kinase or a calmodulin antagonist, but was effectively antagonized by blockers of BK_Ca channels, phosphatidylinositol‐phospholipase C, phosphatidylcholine‐phospholipase C, protein kinase C, SERCA, IP₃‐induced Ca²⁺ release and by pertussis toxin. The ghrelin‐induced increase in the force of contractions was blocked when iberiotoxin (10^?7 mol L^?1) was present in the bath solution. Conclusions: Ghrelin reduces I_K(Ca) in femoral artery myocytes by a mechanism that requires activation of Gα_i/o‐proteins, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C, protein kinase C and IP₃‐induced Ca²⁺ release.     

Excitation-contraction coupling in voltage clamped uterine smooth muscle

1. The relationship between ionic currents and contraction has been investigated in uterine strips of pregnant rat by means of a double sucrose gap apparatus combined with an optical method which permits the measurement of the contraction of the small muscular bundle where potential and current are recorded.2. Effects of duration, size and frequency of imposed potentials upon contraction have been studied. The uterine muscle shows summation and tetanus phenomena. Tension elicited by depolarizing pulses of different durations and amplitudes can be considered as made of two components.3. The first component of the contraction evoked by short depolarizing steps (about 50 ms) depends on the slow inward current. This contraction is abolished by manganese and lanthanum ions and by compound D 600. The amplitude of the tension can be related to the external calcium concentration and consequently to the calcium influx. The slow inward current is supposed to release a part of the bound calcium without excluding, however, a direct activation of myofibrils.4. The second component of the contraction is observed in manganese containing solution with depolarizations longer than 200 ms and without inward current. Such a component of tension suggests the possibility of release of calcium from intracellular stores which could be located in the sarcoplasmic membrane of the uterine smooth muscle.