Epithelium modulates the reactivity of ovalbumin-sensitized guinea-pig airway smooth muscle

Mechanical removal of the airway epithelium alters the in vitro reactivity of airway smooth muscle. The modulation of reactivity may involve the release of inhibitory and excitatory factors from epithelial cells. Guinea pigs sensitized with ovalbumin have been used as an animal model of airway hyperreactivity. We evaluated the influence of the epithelium on the reactivity of in vitro tracheal smooth muscle from control and ovalbumin-sensitized guinea pigs, and the extent to which the presence of the epithelium affects the contractile response to in vitro challenge with ovalbumin. In both control and ovalbumin-sensitized tissues, epithelium removal increased the sensitivity of the preparations to histamine, methacholine and isoproterenol to a similar extent, i.e., 2- to 2.5-fold. Epithelium removal resulted in an 8.1-fold increase in sensitivity to ovalbumin in sensitized tissues. The epithelium appears not only to modulate the reactivity of the tissues to bronchoactive agents, but it also influences the magnitude of the contractile response following antigen challenge.     

Effects of the respiratory epithelium on the tracheal smooth muscle in guinea pigs]

We studied the influence of respiratory epithelium on tracheal smooth muscle tone of guinea pigs. Mechanical removal of the epithelium induced an increase of the contractile response to histamine. In preparation of smooth muscle previously contracted by histamine, isoproterenol induced dose--dependent relaxation, the level of which was significantly greater in tracheal smooth muscle with epithelium than without it. These results suggest an important role of respiratory epithelium on the contractile activity of smooth muscle.     

Bronchial mucosal permeability.

The tracheobronchial epithelium has well-developed tight junctions which on a morphologic basis should be markedly resistant to penetration by protein molecules. Despite this, antigen inhalation in monkeys allergic to Ascaris suum results in the rapid onset of pulmonary physiologic changes. Recent studies in man and animals have shown that a substantial number of mast cells exist in the bronchial lumen and epithelium. We suggest that antigen-antibody interaction initially occurs on these superficial mast cells leading to mediator release and the stimulation of airway irritant receptors. Antigen challenge also results in increased epithelial permeability to protein in the Ascaris-allergic monkey, and from studies on guinea pigs we suggest that this is due to alterations in the tight junctions. Antigen challenge in the monkey also produces increased permeability to labeled histamine and hyperresponsiveness to low concentrations of histamine. We suggest that the apparent airway hyperreactivity to inhaled histamine seen after inhalation of ozone, and NO2, or after upper respiratory infections could be due to damage to epithelial tight junctions. The resultant increase in mucosal permeability would result in an increased amount of histamine reaching airway smooth muscle for a given inhaled concentration.     

Functional identification of epithelial and smooth muscle histamine-dependent relaxing mechanisms in the bovine trachea,but not in bronchi

Theoretically, the overall effect of histamine on respiratory smooth muscle is the result of a subtle balance of contraction and relaxation. The aim of the study was to identify histamine type 2 (H2) and 3 (H3) receptor-dependent relaxing mechanisms in the contractile elements of the bovine tracheobronchial tree. In bronchial preparations, histamine induced very weak contractions, which were not exacerbated with the H2-antagonist cimetidine. Moreover, precontracted bronchial rings never relaxed in response to cumulative doses of histamine or amthamine (H2-agonist). In intact tracheal preparations, histamine induced strong contractions that were exacerbated by cimetidine (E(max): +17.2+/-6.6%) but not by thioperamide (H3-antagonist). Precontracted tracheal bundles did not relax in response to cumulative doses of the H3-agonist R-alpha-methylhistamine. The tracheal contractile response was higher in denuded compared to intact preparations (11.0+/-1.2 vs. 6.0+/-1.7 g). Cimetidine effect was dramatically potentiated in denuded tracheal strips (+40.0+/-11.7%). It is concluded that the weak response of bovine bronchi to histamine is due to a relative scarcity of H1 receptors on bronchial smooth muscle rather than to H2- or H3-dependent relaxation. In the bovine trachea, the smooth muscle possesses relaxing H2 but no H3 receptors. The epithelium exercises a relaxation, which is independent from H2 and H3 receptors.     

Antigen inhalation induces mast cells and eosinophils infiltration in the guinea pig esophageal epithelium involving histamine-mediated pathway

Antigen challenge in sensitized guinea pig esophagus in vitro induces mast cell degranulation and histamine release. This study tests the hypothesis that antigen inhalation in vivo induces infiltration of the esophageal epithelium by mast cells and eosinophils via a histamine pathway. Actively sensitized guinea pigs were exposed to inhaled 0.1% ovalbumin. One or 24 h after inhalation exposure, the esophagus was processed for immunofluorescent staining of mast cell tryptase and eosinophil major basic protein (MBP). Additional animals were pretreated with thioperamide, a histamine H4/H3 receptor antagonist. Total tryptase- and MBP-labeled cells and percent of positive cells in the epithelial layer were counted. The total number of mast cells was unchanged after inhalation challenge, but the percentage in the epithelium increased 1 h after challenge. The total number of eosinophils increased 1 h after challenge, and the percentage migrating to the epithelium increased by 24 h after challenge. Mast cell migration into the mucosal epithelium preceded that of eosinophils. Thioperamide inhibited mast cell and eosinophil migration. In conclusion, antigen inhalation in sensitized animals induces mast cells and eosinophils to infiltrate in the esophageal epithelium via histamine-mediated mechanism.     

The pathophysiology of asthma

Because postmortem studies of humans provide little information on the initial pathophysiologic events in asthma, animal models have been developed. Recently the Ascaris-allergic rhesus monkey has provided an opportunity to examine the onset of pathophysiologic changes following challenge and to correlate them with airway structure. These studies have suggested that the initial interaction between antigen and mast cells may occur in the bronchial lumen or in the epithelium superficial to the tight junctions, where a small but significant percentage of airway mast cells exist. It also appears that this initial antigen-antibody interaction results in the release of mediators that both stimulate the rapidly adapting stretch receptors in the mucosa and alter the mucosal barrier so that proteins of large molecular weight can penetrate. The fact that antigen challenge results in hyperresponsiveness to a subsequent dose of inhaled histamine and increased systemic absorption of histamine suggests that the airway hyperresponsiveness could be related to increased penetration of histamine into the bronchial wall. These observations suggest that the initial event in an acute asthmatic attack is the release of mediators from superficial mast cells, and that this amplifies the allergic response by altering the mucosal permeability so that more antigen reaches the submucosal mast cells. This altered permeability may also help explain the hyperreactivity of the airways to nonspecific airway stimulants in persons with asthma.     

Modulation of the contractile responses of guinea pig isolated tracheal rings after chronic intermittent hypobaric hypoxia with and without cold exposure.

Previous studies have documented that repetitive exposure to intermittent hypoxia, such as that encountered in preparation to high-altitude ascent, influences breathing. However, the impact of intermittent hypoxia on airway smooth muscle has not been explored. Ascents to high altitude, in addition to hypoxia, expose individuals to cold air. The objective of the present study is to examine the effect of chronic intermittent hypobaric hypoxia (CIH) and CIH combined with cold exposure (CIHC) on tracheal smooth muscle responses to various contractile and relaxant agonists. Experiments were performed on tracheal rings harvested from adult guinea pigs exposed either to CIH or CIHC [14 days (6 h/day) at barometric pressure of 350 mmHg with and without cold exposure of 5 degrees C] or to room air (normoxia). CIH and CIHC attenuated maximum contractile responses to ACh compared with normoxia. The maximum contractile response to histamine decreased with CIH, whereas CIHC restored the response back to normoxia. Both CIH and CIHC attenuated maximum contractile responses to 5-HT. Altered contractile responses after CIH and CIHC were independent of epithelium. Isoproterenol-induced relaxation was not altered by CIH, whereas it was enhanced after CIHC, and these responses were independent of the epithelium. The data demonstrate that intermittent exposure to hypoxia profoundly influences contractile response of tracheal smooth muscle, and cold exposure can further modulate the response, implying the importance of cold at high altitude.     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD4, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE2, PGF2 alpha and PGI2 (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD4, methacholine and histamine. LTD4 (3-100 nM), methacholine (0.1-10 microM) or histamine (0.3-30 microM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE2 and PGF2 alpha, but not PGI2, was decreased in tissues lacking epithelium. Indomethacin (1 microM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI2 and is unlikely to be PGF2 alpha or PGE2. However, the possibility remains that the basal release of PGE2 and/or PGF2 alpha derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE2 and PGF2 alpha which may be involved in the maintenance of baseline tone.     

In vitro studies of antigen-induced bronchospasm: effect of antihistamine and SRS-A antagonist on response of sensitized guinea pig and human airways to antigen.

Exposure of sensitized guinea pig tracheal rings or human bronchial strips to specific antigen in vitro resulted in a rapidly developing, prolonged contraction that was resistant to washing. Treatment of the tissue with diphenhydramine, a histamine H1 antagonist, before antigen delayed the onset and decreased the amplitude of the initial phase of the contraction but did not reduce the duration. Diphenhydramine treatment after development of the contraction did not relax the airway tissue. Antigen-induced histamine release from guinea pig trachea and from human bronchus was complete within the initial 15% of the duration of the contraction. Treatment of sensitized airway tissue with FPL 55712, a SRS-A antagonist, before antigen selectively inhibited the prolonged phase of the response. FPL 55712 administration after the development of antigen-induced contraction resulted in relaxation. These data suggest that both histamine and SRS-A are involved in the response of sensitized guinea pig and human airway tissue to antigen, with histamine mediating the early phase of the contraction and SRS-A primarily mediating the protracted phase.     

Pharmacological analysis of reactivity changes in airways due to acute inflammation in cats

Our recent in vitro studies on airways smooth muscles of the cat with turpentine oil inflammation showed the occurrence of a contractile response of tracheal preparations and a significant increase in the isometric tension of lung strips to histamine application. This study was aimed to establish whether histamine H2-receptors participated in the changed in vitro reactivity of the airways smooth muscles of cats suffering from experimentally induced airway inflammation. Pretreatment of control tracheal preparations, control and experimental groups of the lung strips by cimetidine did not change the character of the histamine response. Similarly, the amplitude of histamine relaxation, of the tracheal preparations partially contracted by carbachol was unchanged by experimental inflammation. Clemastine significantly shifted the histamine dose-response curves to the right in both groups of lung strips. However, significant differences in lung strip reactivity between control and experimental groups of cats were not eliminated. Our results do not support the role of histamine H2-receptors in the pathologically increased airway reactivity to histamine in vitro.     

O3-induced airway hyperresponsiveness to noncholinergic system and other stimuli.

The effect of O3 exposure (3 ppm, 1 h) on the in vivo and in vitro airway responsiveness, as well as the changes in cell contents in bronchoalveolar lavage (BAL) fluid, were evaluated 16-18 h after O3 exposure in sensitized and nonsensitized male guinea pigs. The sensitization procedure was performed through repeated inhalation of ovalbumin for 3 wk. Increase in pulmonary insufflation pressure produced by the excitatory nonadrenergic noncholinergic (eNANC) system, histamine, and antigen were assessed in in vivo conditions, whereas airway responsiveness to histamine and substance P was evaluated in in vitro conditions by use of tracheal chains with or without epithelium and lung parenchymal strips. We found that O3 exposure 1) increased the neutrophil content in BAL fluids in both sensitized and nonsensitized guinea pigs, 2) caused hyperresponsiveness to eNANC stimulation in nonsensitized guinea pigs (although combination of sensitization and O3 exposure paradoxically abolished the hyperresponsiveness to eNANC stimulation), 3) increased the in vivo bronchoconstrictor responses to histamine and antigen, 4) caused hyperresponsiveness to substance P in nonsensitized tracheae with or without epithelium and in sensitized tracheae with epithelium, 5) did not modify the responsiveness to histamine in tracheae with or without epithelium (and in addition, epithelium removal caused hyperresponsiveness to histamine even in those tracheae exposed to O3), and 6) produced hyperresponsiveness to histamine in lung parenchymal strips either from sensitized or nonsensitized guinea pigs.     

Histamine, endogenous prostaglandins and cyclic nucleotides in the regulation of airway muscle responses in the guinea pig

Indomethacin (30 mg/kg, i.p.) reduced pulmonary resistance in guinea pigs but did not affect their sensitivity to histamine. This treatment preferentially reduced the generation of PGE2 by isolated tracheal preparations. The ratios of PGF2 alpha/PGE2 before and after treatment were 1/1 and 6/1, respectively. Chronic indomethacin treatment (30 mg/kg, i.p., twice a day for 4 days) increased histamine sensitivity in vivo 2 fold while a longer treatment (10 days) was without effect. The efficacy of histamine and the potency of isoproterenol in tracheal tissues were unaffected by either treatment. Indomethacin (17 microM for 30 min) relaxed tracheal tissues but not bronchial tissues. Responses of both tissues to contractile agonists were potentiated after indomethacin treatment. The efficacy of histamine was smaller in bronchi than in tracheas. Similarly, PGE2, PGI2 and isoproterenol were less potent in bronchi. Basal amounts of cyclic AMP were higher in bronchi than in tracheas; indomethacin did not affect the basal amounts of cyclic AMP in tracheal tissues but reduced them in bronchial preparations. Histamine elevated cyclic AMP content in both preparations; this elevation was reduced by indomethacin. While prostaglandins play a role in modulating airway responses in vitro, their role in airways in normal animals in vivo is more difficult to demonstrate.     

An indomethacin-sensitive contraction induced by beta-antagonists in guinea pig airways

Beta-adrenergic receptor (beta-AR) antagonists have been associated with increased airway reactivity in asthmatics and potentiation of contractile stimuli in animal models. In the present study, using an in vitro model of tracheal preparations from guinea pigs, we show that the beta-AR antagonists propranolol and pindolol induce a smooth muscle contraction. A prerequisite for this contraction is that the airway preparations have been pre-treated with an beta-AR agonist. Our data show that the contractile effect of beta-AR antagonists is not a simple consequence of reversing the agonist-induced relaxation. Furthermore, the effect seems to be mediated through interaction with beta2-ARs since the response is stereo-selective, and the selective beta1-AR receptor antagonist atenolol did not induce any contractile response. SQ 29,546, a thromboxane A2 antagonist; MK 886, a lipoxygenase inhibitor; and indomethacin, a cyclooxygenase inhibitor significantly inhibited the contractions of the tracheal preparations induced with propranolol or pindolol. We put forward the hypothesis that the contractile effect of the beta-AR antagonist is a consequence of their inverse agonist activity, which is only evident when the receptor population have a higher basal activity. Our results indicate a novel additional explanation for the known side effect, bronchoconstriction, of beta-AR antagonist.     

The effect of synthetic leukotrienes on tracheal microvascular permeability

The effect of synthetic leukotrienes (LT) C4, D4 and E4 on the permeability of the airway microvasculature to plasma albumin was quantitatively evaluated using an in situ guinea pig tracheal model. Vascular permeability was measured as extravascular albumin content by employing 125I-bovine serum albumin and, in order to correct for blood volume, 51Cr-erythrocytes were used. Intratracheal injection of synthetic LTC4, LTD4 and LTE4 (0.1-1000 ng) produced dose-dependent increases in tracheal extravascular albumin content. The leukotrienes were approximately 100-1000 fold more potent than histamine, although histamine did produce a greater maximal increase in extravascular albumin than the leukotrienes. Methacholine did not increase extravascular albumin content. The microvascular permeability effect of LTD4 was antagonized by FPL 55712 but not by mepyramine; conversely, the effect of histamine was antagonized by mepyramine and not by FPL 55712. Additionally, indomethacin did not alter the LTD4-induced increases in tracheal vascular permeability. These results suggest that the effect of LTD4 on tracheal microvascular permeability is directly mediated and is not the indirect result of cholinergic stimulation, histamine release or de novo synthesis of cyclooxygenase products.     

CLA reduces antigen-induced histamine and PGE(2) release from sensitized guinea pig tracheae

Conjugated linoleic acid (CLA) has been shown to enhance immune reactions such as lymphocyte blastogenesis and delayed-type hypersensitivity. We investigated the role of CLA in type I (immediate) hypersensitivity, using a guinea pig tracheal superfusion model for measuring antigen-induced airway smooth muscle contraction and inflammatory mediator release. Female Hartley guinea pigs were fed a diet supplemented with 0.25 g corn oil or linoleic acid/100 g of diet (control) or 0.25 g CLA/100 g of diet for at least 1 wk before and during active sensitization to ovalbumin antigen. Tracheae from sensitized guinea pigs were suspended in air-filled water-jacketed (37 degrees C) tissue chambers in a superfusion apparatus. Tracheae were superfused with buffer containing antigen, and tissue contraction was recorded. Superfusate was collected at 90-s intervals for evaluation of histamine and PGE(2) release. CLA did not affect antigen-induced tracheal contractions when expressed as gram contraction per gram tissue. CLA significantly reduced antigen-induced histamine and PGE(2) release. CLA appears to decrease release of some inflammatory mediators during type I hypersensitivity reactions.     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD₄, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE_2α and PGI₂ (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD₄, methacholine and histamine. LTD₄ (3–100 nM), methacoline (0.1–10 μM) or histamine (0.3–30 μM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE₂ and PGF_2α but not PGI₂, was decreased in tissues lacking epithelium. Indomethacin (1 μM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI₂ and is unlikely to be PGF_2α or PGE₂. However, the possibility remains that the basal release of PGE₂ and/or PGF_2α derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE₂ and PGF_2α which may be involved in the maintenance of baseline tone.     

Release of leukotrienes from guinea pig lung stimulated by C5ades Arg anaphylatoxin

The complement anaphylatoxins C5a and C5Ades Arg contract guinea pig peripheral airway preparations and trachea by a mechanism largely independent of histamine release. In trachea the contractions are inhibited by FPL 55712, a relatively specific inhibitor of slow-reacting substance of anaphylaxis (SRS-A). SRS-A is now known to be a mixture of leukotrienes C4, D4, and E4 (LTC4, LTD4, LTE4). These data suggest that C5-derived anaphylatoxins stimulate production and release of leukotrienes in pulmonary tissues. To define these observations more precisely, fragments of guinea pig lung were incubated with porcine C5ades Arg, and the supernatant fluids were analyzed for leukotrienes by using both pharmacologic and chemical methods. In addition to histamine, a smooth muscle contracting activity characteristic of SRS-A was released from C5a-treated lung preparations. The contractile substance was identified as a leukotriene based on: 1) the characteristic contraction of guinea pig ileum, 2) inhibition of the contractile activity by FPL 55712, 3) enhanced release of activity in the presence of indomethacin or L-cysteine, 4) chromatographic behavior of ethanol-extracted active material on Amberlite XAD-7 resin, and 5) cochromatography of the active material on reverse-phase, high performance liquid chromatography with standard LTD4. We therefore concluded the humoral factor C5ades Arg induces a leukotriene release reaction in guinea pig lung tissue. This particular response of pulmonary tissue to anaphylatoxin has not been appreciated previously as an immediate effect of complement activation on the pathophysiology of the lung.     

Reduced anaphylactic responsiveness of strain 2 guinea pigs.

Strain 2 guinea pigs have been shown to have diminished anaphylactic responsiveness. In the present study, experiments were conducted comparing various characteristics of the anaphylaxis-resistant Strain 2 guinea pigs to those of an outbred anaphylaxis-prone Dunkin-Hartley strain. To bypass the possibility that differences in antibody titers accounted for the difference in anaphylactic reactivity, both strains of guinea pig were passively sensitized with the same amount of IgG antibody to ovalbumin. Measures of anaphylactic responsiveness to subsequent antigen challenge with ovalbumin included (i) systemically induced respiratory responses; (ii) isolated cardiac responses; and (iii) cutaneous responses. In all cases, using an amount of antibody sufficient to sensitize Dunkin-Hartley guinea pigs, the anaphylactic responses of the Strain 2 guinea pigs were either nonexistent or significantly less than those of the Dunkin-Hartley strain. To further determine which factors might be responsible for this difference, tissue histamine content, histamine releasability, and histamine responsiveness of the two strains were measured. The results of these studies indicated that the respiratory hyporesponsiveness of the Strain 2 guinea pigs may be due to a low pulmonary histamine content combined with reduced pulmonary responsiveness to histamine. However, since the cardiac histamine content and the responsiveness of the Strain 2 guinea pigs were not different from those of the Dunkin-Hartley strain, these factors cannot contribute to the reduced Strain 2 cardiac anaphylactic responsiveness. Compound 48/80 released equal quantities of histamine from the isolated hearts of the Strain 2 and the Dunkin-Hartley animals, but antigen challenge evoked histamine release only from the isolated Dunkin-Hartley hearts. We conclude that the cardiac anaphylactic hyporesponsiveness of the Strain 2 guinea pigs may be due to an inability of antigen to evoke release of anaphylactic mediators such as histamine.     

Reduction in basal adenylate cyclase activity during the immunologic release of histamine from guinea pig lung.

Cyclic AMP has been implicated in the regulation of the immunologic release of histamine from lung and other tissues and cell types. The mechanism whereby intracellular levels of cAMP are altered during mediator release was investigated. Measurements of histamine, adenylate cyclase, and cAMP phosphodiesterase activities were made in actively and passively sensitized guinea pig lung after challenge with antigen. A transient decrease in basal adenylate cyclase activity occurred which returned to control levels after histamine release. There was no change in cAMP phosphodiesterase activity determined at substrate concentrations of 1 mM and 0.01 mM. The adenylate cyclase response did not occur under the following conditions: 1) incubation of nonsensitized lung with antigen, 2) incubation of sensitized lung with antigen in the absence of extracellular calcium, and 3) incubation of nonsensitized lung with compound 48/80. These observations indicate 1) the adenylate cyclase response and the immunologic release of histamine are intimately related, and 2) the reduction in intracellular levels of cAMP which have been reported to occur during immunologic histamine release are mediated via adenylate cyclase.     

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.