We report the development of a nontransformed line of human airway smooth muscle cells retaining smooth muscle-specific contractile protein expression and physiological responsiveness to agonists implicated in inflammatory airway diseases. Specific responses to histamine, leukotrienes, bradykinin, platelet-activating factor, substance P, and thromboxane analogues are demonstrated as well as functional coupling to beta-adrenergic receptors. The cell line was characterized using indirect immunofluorescence, as well as electrophoretic separation and immunoblot analysis of smooth muscle-specific actin. Functional responses were assessed by measurements of cytosolic calcium and stimulation of adenosine 3',5'-cyclic monophosphate production. The cells retain their responsiveness over many population doublings and should be a useful model to examine specific receptor-effector mechanisms, as well as the effects of neurohumoral agents on the regulation of airway smooth muscle growth and differentiation.

A human airway smooth muscle cell line that retains physiological responsiveness.

Electroporation is described mathematically by a partial differential equation (PDE) that governs the distribution of pores as a function of their radius and time. This PDE does not have an analytical solution and, because of the presence of disparate spatial and temporal scales, numerical solutions are hard to obtain. These difficulties limit the application of the PDE only to experimental setups with a uniformly polarized membrane. This study performs a rigorous, asymptotic reduction of the PDE to an ordinary differential equation (ODE) that describes the dynamics of the pore density $N(t).$ Given $N(t),$ the precise distribution of the pores in the space of their radii can be determined by an asymptotic approximation. Thus, the asymptotic ODE represents most of the phenomenology contained in the PDE. It is easy to solve numerically, which makes it a powerful tool to study electroporation in experimental setups with significant spatial dependence, such vesicles or cells in an external field.

Asymptotic model of electroporation

Cell electropermeabilization: a new tool for biochemical and pharmacological studies.

Abundant data from animal models and humans support the hypothesis that changes at the level of parasympathetic neuronal control of airway smooth muscle result in increased bronchoconstriction in response to vagal stimulation, leading to airway hyperresponsiveness. Neuronal inhibitory M2 muscarinic acetylcholine receptors on parasympathetic nerves are responsible for limiting acetylcholine release from these nerves. In humans with asthma, and after pulmonary inflammatory events in experimental animals, these receptors are dysfunctional, which results in airway hyperresponsiveness. Although it is unknown what mechanisms underlie airway hyperresponsiveness in chronic obstructive pulmonary disease, loss of parasympathetic control of airway smooth muscle is thought to be a contributing mechanism. As such, anticholinergic therapy is used extensively and with a high degree of success in the treatment of this condition. The future for inhaled anticholinergic compounds for the treatment of chronic obstructive pul...

https://www.atsjournals.org/doi/pdf/10.1513/pats.200504-043SR

Cholinergic pathways in the lungs and anticholinergic therapy for chronic obstructive pulmonary disease.

BACKGROUND: The phrenic nerve can be injured with radiofrequency energy delivery. Nevertheless, the mechanisms of injury are unknown. This study was undertaken to examine phrenic nerve tissue temperatures during ablation at the pulmonary vein (PV) orifice, assess the temperature dependence of injury, and to delineate the possible mechanisms of untoward nerve effects. METHODS: Ten dogs underwent ablation at the right superior PV (RSPV) orifice. Phrenic nerve temperatures were assessed with implanted thermocouples overlying the endocardial ablation site. Using an 8-mm ablation catheter tip, energy was titrated to 50 degrees C and incremented by 5 degrees C for 120 seconds. RESULTS: Phrenic nerve capture was achieved in nine (90%) dogs after thermocouple implantation. A RSPV orifice tissue temperature >60 degrees C occurred in 32 (84%) of energy deliveries with a power of 34 +/- 22 W. In three (33%) dogs, this resulted in nerve dysfunction (maximum nerve temperature: 41 degrees C, 41 degrees C, and 91 degrees C) with histology consistent with acute thermal injury. In four additional dogs, 17 energy deliveries were made directly to the phrenic nerve using a novel in situ model. In 5 (29%) energy deliveries, nerve function was impacted immediately by the generated current, with resolution simultaneous with discontinuing radiofrequency. Transient phrenic nerve injury occurred in all dogs at a temperature of 47 +/- 3 degrees C (range: 43-53 degrees C) after 38 +/- 32 seconds (range: 20-120 seconds). After termination of the energy delivery, nerve function returned in 15(88%) during 30 seconds of postablation pacing. In two (12%) ablation attempts, nerve recovery was delayed (>3 minutes). Permanent injury occurred in all dogs after 92 +/- 83 seconds (range: 20-280 seconds) of additional energy delivery at a temperature of 51 +/- 6 degrees C (range: 45-65 degrees C). CONCLUSION: Phrenic nerve injury can be more common than anticipated with RF ablation at the RSPV orifice. Relatively low tissue temperatures can injure the nerve. Immediate nerve effects suggest a second mechanism of nerve dysfunction related to electrical current. Transient nerve effects occur prior to permanent damage, providing an opportunity to discontinue energy delivery before permanent injury.

Mechanisms of phrenic nerve injury during radiofrequency ablation at the pulmonary vein orifice.

The goal of our study was to test for an inhibitory effect of acetylcholine on adenylate cyclase activity in canine trachealis muscle. Therefore, cells were dispersed from the muscle enzymatically and lysed, and adenylate cyclase activity was assayed in a membrane suspension isolated from the lysates. Maximal beta-adrenergic stimulation, in the presence of GTP (10(-4) M), increased the activity of adenylate cyclase twofold above the activity induced by GTP alone. In the presence of GTP, acetylcholine (10(-4) M) decreased activity from 97 +/- 21 to 55 +/- 13 pmol cyclic AMP X min-1 X mg protein-1 (means +/- SE; n = 5; P less than 0.05); in the presence of GTP plus isoproterenol (10(-4) M), the acetylcholine-induced decreases were from 163 +/- 29 to 101 +/- 15 pmol cyclic AMP X min-1 X mg protein-1 (P less than 0.05). These decreases were dose dependent and they were altered by a series of cholinergic agents in a pattern consistent with a muscarinic effect. Our results suggest that one biochemical effect of vagal stimulation in the central airways of dogs may be attenuated adenylate cyclase activity in the smooth muscle.

Muscarinic cholinergic inhibition of adenylate cyclase in airway smooth muscle.

Muscarinic agonists are potent constrictors of airway smooth muscle. In many tissues, muscarinic agonists also reduce intracellular cyclic AMP by inhibiting its synthesis. In airway smooth muscle, the role muscarinic agonists have in the regulation of cyclic AMP content is not established. The hypothesis of our study was that muscarinic agonists reduce cyclic AMP accumulation in dog tracheal smooth muscle, and that this reduction involves a pertussis toxin-sensitive regulatory protein (Gi) that couples occupancy of the muscarinic receptor by the agonist to inhibition of adenylate cyclase. We measured cyclic AMP accumulation in tracheal smooth muscle from 4 dogs, and found that acetylcholine (10−4 M) diminished basal and isoproterenol-stimulated cyclic AMP accumulation by 37.6 ± 12.1% and 39.4 ± 1.9%, respectively (mean ± SEM, p < 0.05). This reduction of cyclic AMP was dose-dependent and inhibited by atropine (10−5 M). Incubation of dog tracheal smooth muscle with pertussis toxin (12.5 µg/ml) for 21 h cat...

Muscarinic cholinergic inhibition of cyclic AMP accumulation in airway smooth muscle. Role of a pertussis toxin-sensitive protein.

The goals of our study were to isolate smooth muscle cells from the trachealis muscle of adult dogs and to characterize the cells morphologically when they were maintained in primary culture Enzymatic digestion of the muscle yielded 48 +/- 18 X 10(6) viable smooth muscle cells per gram of tissue When placed in culture, these cells rapidly proliferated until confluence was reached The proliferating cells in culture differed from the cells in the intact tissue in that they stained less intensely for smooth muscle myosin, developed immunofluorescent staining for the intermediate filament protein vimentin, and lost many of the ultrastructural properties of the intact muscle Only within nodules of cells in the confluent cultures were these ultrastructural properties preserved Cultures of canine tracheal fibroblasts differed from these smooth muscle cell cultures in that the fibroblasts did not stain for smooth muscle myosin and did not form nodules at confluence We concluded that adult canine airway smooth muscle cells may be maintained in primary culture, that the confluent cultures contain nodules of cells with many morphologic characteristics of the intact muscle, and that these preparations may be distinguished from cultured canine tracheal fibroblasts on specific morphologic grounds

Morphologic characterization of cultured smooth muscle cells isolated from the tracheas of adult dogs

Muscarinic cholinergic receptors have been classified into subtypes based on their high (M-1 subtype) or low (M-2 subtype) affinities for the nonclassic antagonist pirenzepine, and this classification has important experimental and therapeutic implications. Because muscarinic receptors are abundant in the airways where they mediate several different cellular responses, the goal of this study was to characterize the affinities of pirenzepine for the muscarinic receptors in bovine tracheal mucosa and smooth muscle. After isolating membrane particulates from mucosa and smooth muscle, as well as from bovine cerebral cortex (a known source of M-1 receptors), we used /sup 3/H-quinuclidinyl benzilate to label muscarinic receptors in the particulates and performed competition radioligand binding assays in the presence of either atropine or pirenzepine. Receptors from all 3 tissues (mucosa, smooth muscle, and cerebral cortex) were of a relatively uniform affinity for atropine (range of KI values: 0.8 +/- 0.4 X 10(-9) to 2.4 +/- 1.7 X 10(-9) M), as would be predicted for this classic muscarinic antagonist. By contrast, affinities for pirenzepine differed depending on the tissue. In cerebral cortex, the majority of receptors were of high affinity for pirenzepine (KI = 1.8 +/- 1.4 X 10(-8) M). In both mucosa and smooth muscle,more » receptors were of low affinity for pirenzepine (Kl = 4.8 +/- 0.4 to 6.9 +/- 3.8 X 10(-7) M). We conclude that muscarinic cholinergic receptors in bovine tracheal mucosa and smooth muscle are predominantly of the M-2 subtype.« less

Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle.

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3′,5′-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.

J. K. Brown

Papers

Muscarinic cholinergic inhibition of adenylate cyclase in airway smooth muscle.

Muscarinic cholinergic inhibition of cyclic AMP accumulation in airway smooth muscle. Role of a pertussis toxin-sensitive protein.

Morphologic characterization of cultured smooth muscle cells isolated from the tracheas of adult dogs

Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle.

Differential effects of prostaglandin E2 on contractions of airway smooth muscle.