Electrical behavior of guinea pig tracheal smooth muscle

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of -50 +/- 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca(2+) channels with nifedipine or by depleting intracellular Ca(2+) stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 microM). In quiescent cells, nifedipine (1 microM) reduced EJPs by 30%, whereas ryanodine (10 microM) reduced EJPs by 93%. These results suggest that both the release of Ca(2+) from intracellular stores and the influx of Ca(2+) through voltage-dependent Ca(2+) channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.     

Noncholinergic contractile response to nicotine in the guinea pig isolated tracheal smooth muscle

The existence of substance P immunoreactive nerves in the trachea of guinea pig is known. In this study, capsaicin induced a long-lasting and marked contraction in the guinea pig trachea and nicotine-induced contraction was partially reduced in the capsaicin-treated muscle. Furthermore, the contractile response to nicotine (10(-5) M) in the presence of atropine (10(-7) M) was abolished by a substance P antagonist, [D-Arg1, D-Pro2, D-Trp7,9 Leu11]substance P (10(-5) M). These findings suggest that noncholinergic contractile response to nicotine may be due to the release of material(s) resembling substance P in the isolated tracheal smooth muscle preparation of guinea pig.     

Studies of mediator involvement in cooling-induced alterations of guinea pig tracheal smooth muscle contractility

Heat loss from airway smooth muscle is a potent stimulus which causes substantial, but poorly understood, alterations in muscle tension. This study considered the involvement of endogenous mediators in cooling-induced tension changes in incubated guinea pig trachea. Smooth muscle tension was monitored in tracheal cylinders which were carefully cooled from 37 to 30 degrees C in the presence or absence of various inotropic mediators. In our study, cooling alone, at a rate of 1 degree C/min, was associated with an average loss of smooth muscle tension of 88.2 mg. Cooling tracheal tissue that had been previously exposed to 3 X 10(-6) M histamine, however, caused an additional increase in tracheal tension of 133 mg, over and above that caused by histamine alone. In the presence of 10(-5) M prostaglandin F2 alpha, or 10(-5) M thromboxane B2, cooling was associated with respective losses of smooth muscle tension of 211.4 and 211.2 mg, as compared to the tension associated with these mediators when they were used alone under control conditions. When the speed of tracheal cooling was increased to 40 degrees C/min, there was a slight increase in tension for 20 sec followed by a pronounced and sustained relaxation. The mechanisms involved in the response of airway smooth muscle to cooling are complex. The results of our study, however, suggest that mediators may play a role in the cooling-induced alterations of airway smooth muscle tension.     

Characteristics of beta-adrenoceptors in tracheal smooth muscle of guinea pig sensitized by egg albumin

The effect of pretreatment with egg albumin was examined on the beta-adrenoceptors in guinea pig isolated trachea. Befunolol and carteolol acted as partial agonists and their pA2 values were significantly larger than their corresponding pD2 values in tracheae from both untreated guinea pigs and those treated with egg albumin, suggesting that the beta-adrenoceptors contain two different affinity sites. The Scatchard plot of specific [3H]befunolol binding showed two affinity sites of the receptor (high and low affinity sites) in tracheae from both untreated animals and those treated with egg albumin. The pKD values of befunolol for both low and high affinity sites were in agreement with their respective pD2 and pA2 values. The intrinsic activities of befunolol and carteolol and the pD2 values of the test drugs were decreased by the treatment with egg albumin. The treatment with egg albumin also decreased the total amount of the two affinity sites of the receptor without any change in affinity. The present results support the partial blockade of beta-adrenoceptors in asthma proposed by Szentivanyi.     

Induction of rhythmic contraction in canine tracheal smooth muscle

Na(+)-K+ ATPase activity of the canine tracheal smooth muscle membrane is responsible for the electrogenic pumping of Na+ and K+ ions. It has been shown that this activity results in muscle relaxation. Based on the results of the current study, we suggest that prolonged electrical stimulation induces increased Na(+)-K+ ATPase activity in isolated tracheal smooth muscle. Tracheal smooth muscle pretreated with prolonged electrical stimulation developed graded mechanical activity when subsequently treated with histamine, serotonin, acetylcholine, or 80 mM K+. This increased isometric tension was interrupted by rhythmic activity, which was elicited by histamine or serotonin but not by acetylcholine or 80 mM K+ stimulation. The spontaneous phasic activity was not inhibited by atropine or propranolol but was totally inhibited by 10(-6) M ouabain. These results suggested that the relaxation phase of rhythmic contraction in response to histamine and serotonin stimulation could be the result of stimulated Na(+)-K+ ATPase activity.     

Two components of contraction in guinea pig papillary muscle

Biphasic contractions were produced in guinea pig papillary muscle by inducing partial depolarization in a K+ -rich solution (22 mM) containing 10(-6) M isoproterenol. However, when the same conditions were applied to frog and rat, monophasic contractions were obtained. In the case of guinea pig, an increase in the beating frequency produced an increase in amplitude of the first component and a reduction of the second, while in frog and rat, only a decrease in the amplitude of contractions was recorded. Caffeine (10(-3) M) eliminated the first component and increased the second in guinea pig, while in the case of rat and frog it decreased the amplitude of contractions. Procaine (10(-3) M) suppressed the first component and decreased the second one. The contraction in frog appears to be similar to the second component of contraction in guinea pig, while in rat, the contraction is comparable with the first component in guinea pig. It is suggested that the calcium ions which activate the two components of contraction in guinea pig under the given experimental conditions may arise from two different sources.     

Vagal innervation of guinea pig bronchial smooth muscle

We isolated the guinea pig right bronchus with the vagus nerves intact and evaluated the changes in isometric tension of the smooth muscle in response to nerve stimulation. Brief (10-s) trains of electrical field stimulation or vagus nerve stimulation caused a biphasic contraction: the "first phase" sensitive to atropine and the "second phase" sensitive to capsaicin. The two phases could be dissociated by adjusting the stimulus intensity; greater stimulus intensities (pulse durations or voltage) were required to evoke the capsaicin-sensitive phase. When stimulated at 30-min intervals, the magnitude of both phases of the contractions declined over a 2-h period of repeated stimulation; however, this was prevented by indomethacin. Stimulation of the left vagus nerve resulted in a monophasic contraction of the right bronchus, with little evidence of a capsaicin-sensitive phase. Blocking neurotransmission through the bronchial ganglion, as monitored by intracellular recording techniques, abolished the first-phase contraction but had no effect on the capsaicin-sensitive phase. Selective blockade of muscarinic M1 receptors had no effect on vagus nerve-mediated contractions. The results demonstrate that the left and right vagus nerves carry preganglionic fibers to the right bronchial ganglion. The right but not the left vagus nerve also carries capsaicin-sensitive afferent fibers that, when stimulated, result in a persistent contraction of the right bronchus. Finally, we provide functional and electrophysiological evidence supporting the hypothesis that capsaicin-sensitive afferent neurons communicate with postganglionic motoneurons within the bronchus.     

Components of cholinergic excitation coupling with smooth muscle contraction in the guinea pig taenia coli]

In concentrations of 10(-9)-10(-7) g/ml acetylcholine increased the tone of the smooth muscles of the longitudinal band of the large intestine of a guinea pig, increasing the permeability of the cellular membranes for the entering flux of 45Ca2+. In concentrations of 10(-6) g/ml and over acetylcholine caused a release of the membranous calcium and in the concentrations of 10(-5)-10(-3) g/ml markedly increased the permeability of the membranes of the smooth muscle cells for the 22Na+ ions causing depolarization and an increase in the frequency of the action potentials. It is supposed that the coupling of the cholinergic stimulus with the end effect (muscle contraction) included 3 components: intensification of the entrance of Ca2+ into the smooth muscle cells, release of the membrane calcium and adhesion mechanism.     

Endogenous prostaglandins modulate histamine-induced contraction in canine tracheal smooth muscle

We studied the role of endogenous prostaglandins in modulating the histamine response of canine tracheal smooth muscle (TSM) in vitro. Indomethacin (INDO) (10(-7) - 10(-5) M), a cyclooxygenase and prostaglandin synthesis inhibitor, significantly increased maximum histamine-induced tension (Tmax) and decreased the concentration of histamine required to produce 50% of Tmax (EC50). Acetylsalicylic acid (10(-5) -5 X 10(-4) M), another less potent cyclooxygenase inhibitor, also decreased EC50. Neither the lipoxygenase inhibitor nordihydroguaiaretic acid nor the leukotriene antagonist FPL 55712 had any effect on histamine-induced tension in INDO-pretreated TSM. INDO reduced the standard deviation of EC50 from 0.47 in control TSM (n = 51) to 0.26 in INDO-pretreated TSM (n = 31) (P less than 0.02). High-pressure liquid chromatography established prostacyclin (PGI2), through its degradation product 6-oxo-PGF1 alpha, as the predominant prostaglandin produced by canine TSM. Exogenous PGI2 caused a concentration-dependent relaxation of histamine-contracted TSM. In the tissue bath, spontaneous efflux of 6-oxo-PGF1 alpha from TSM, as measured by radioimmunoassay, averaged 4.7 ng . g muscle-1 . min-1 and increased to 10 ng/g muscle (n = 10, P less than 0.001) with administration of histamine. The isometric tension produced by histamine (10(-4) M) was inversely linearly correlated with the log concentration of endogenous 6-oxo-PGF1 alpha (r = 0.81, P less than 0.01). Our results are consistent with an important role for endogenous bronchodilating prostaglandins, probably prostacyclin, in determining both the histamine sensitivity of canine TSM in vitro and its variability among individual animals.     

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

The influence of L-NG-nitro-arginine on field stimulation induced contractions and acetylcholine release in guinea pig isolated tracheal smooth muscle

The interaction between parasympathetic and inhibitory non-adrenergic, non-cholinergic nerves in tracheal smooth muscle was investigated by determining the effects of the NO-synthase inhibitor L-NG-nitro-arginine (L-NOARG) on contractions and the associated acetylcholine release elicited by field stimulation of the muscle. At frequencies above 2Hz contractile responses to field stimulation were potentiated by L-NOARG (50 microM). alpha-chymotrypsin pre-treatment potentiated contractile responses at all frequencies, but the effects of L-NOARG were unaltered. The effect of L-NOARG on responses to 5Hz electrical stimulation was not mimicked by D-NOARG, was reversed by L-, but not D-arginine and was unaffected by epithelium removal. L-NOARG did not affect responses to exogenous acetylcholine nor the overflow of 3H from tissues previously loaded with [3H]-choline. It is therefore concluded that field stimulation of tracheal smooth muscle induces the release of an endogenous nitrate, which, by an inhibitory action on smooth muscle, functionally antagonises the concomitantly released parasympathetic neurotransmitter.     

Increased in vitro responses of tracheal smooth muscle from hyperresponsive guinea pigs

Repeated aerosol antigen challenge of previously sensitized guinea pigs induces airway hyperresponsiveness to inhaled acetylcholine. To determine the mechanism producing these airway changes and assuming that changes in the trachealis muscle reflect changes in muscle of the entire tracheobronchial tree, we examined the in vitro smooth muscle mechanics and morphometric parameters of tracheae from guinea pigs demonstrating hyperresponsiveness in vivo vs. tracheae from control guinea pigs. No differences between these groups were found in luminal volume at zero transmural pressure, passive pressure-volume characteristics, or area of airway wall. Smooth muscle areas were slightly less in tracheae from hyperresponsive guinea pigs. Tracheae from hyperresponsive guinea pigs had both significantly increased isovolumetric force generation and isobaric shortening compared with tracheae from controls when evaluated over the range of transmural pressures from -40 to 40 cmH2O. We conclude that the in vivo airway hyperresponsiveness induced with repeated antigen challenge is associated with both increased force generation and shortening of tracheal smooth muscle without increased muscle mass, suggesting enhanced contractile activity.     

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Intracellular Ca(2+) waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca(2+) and voltage recordings with microelectrodes, respectively. Spontaneous Ca(2+) waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca(2+)-induced Ca(2+) release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca(2+) waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca(2+) waves were abolished by the Ins(1,4,5)P(3) receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca(2+) waves and induced Ca(2+) flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca(2+) waves depend on sarco(endo)plasmic reticulum (SR) Ca(2+) release via Ins(1,4,5)P(3) receptors, PLC activity, Ca(2+) influx via VDCCs, and SR Ca(2+) concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca(2+) release via Ins(1,4,5)P(3) receptors. Ca(2+) waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.     

Responsiveness of isolated tracheal smooth muscle from normal and sensitized guinea pigs

Studies of the responsiveness of strips of tracheal smooth muscle and the changes after sensitization of ovalbumin were carried out. The hypothesis that there might be a generalized or a selective change of airway smooth muscle responsiveness to sensitization was examined in vitro. Agonists acting on muscarinic receptors, alpha 1-, alpha 2-, and beta-adrenoceptors, purine receptors, histamine and serotonin receptors, and leukotriene and prostaglandin receptors were tested, as well as mediators released from local nerves by field stimulation and procedures such as elevation of potassium or addition of Ca2+ ionophores which do not involve specific receptors. Sensitivity to serotonin increased significantly in sensitized animals. Total magnitude of the contraction and subsequent relaxation responses to field stimulation also increased significantly. Neither of these changes was large in magnitude. Although there were a few minor changes in sensitivity (pD2) or in maximum responses, the hypothesis of important changes in responses of any sort in tracheal muscle after sensitization was rejected. The question was raised whether this general absence of changed responsiveness in vitro reflected (i) the failure of sensitization to induce generalized smooth muscle hyperresponsiveness, (ii) the loss of the mechanisms of such responsiveness in vitro, or (iii) the inadequacy of in vitro techniques to assess responsiveness present in vivo.