Introduction

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Serotonin or 5-hydroxytryptamine (5-HT) is synthesized by pulmonary neuroendocrine cells located in the airway mucosa and is released in response to changes in the gaseous composition of the airway lumen (Lauweryns et al., 1973, 1977). 5-HT has long been recognized as a bronchoconstrictor in many species, acting through direct activation of serotonergic receptors on airway smooth muscle (ASM) and indirectly through prejunctional modulation of cholinergic and peptidergic neurotransmitter release (Hahn et al., 1978; Buckner et al., 1991). 5-HT, however, does not contract ASM of all species, and in some, such as humans, it may actually produce relaxation (Goldie et al., 1982). In guinea pig isolated airways, 5-HT induces contraction or relaxation, depending on the concentration and the level of preexisting tone on the muscle (Bayol et al., 1985; Baumgartner et al., 1990; D'Agostino et al., 1996). The cellular mechanism for 5-HT-induced contraction of ASM has been well characterized and is similar to that of other contractile receptor agonists. 5-HT interacts with specific receptors to stimulate inositol phosphate metabolism, Ca²⁺ mobilization, and protein kinase C activation (Baumgartner et al., 1990; Yang et al., 1994a,b; Watts et al., 1994; Tolloczko et al., 1995). In contrast, the mechanism by which 5-HT relaxes ASM is unknown.

The Na⁺-K⁺ pump is a ubiquitous membrane protein that catalyzes the active transport of K⁺ into cells and Na⁺ out of cells against their electrochemical gradient. Na⁺-K⁺ pump activity is regulated by a variety of hormones, neurotransmitters, and growth factors. 5-HT, in particular, activates the Na⁺-K⁺ pump in the brain (Hernandez, 1992), kidney (Soares-da-Silva et al., 1996), and vascular smooth muscle (Navran et al., 1991). Indeed, stimulation of the Na⁺-K⁺ pump by 5-HT has been proposed to mediate the inhibitory effect that 5-HT exerts on vascular smooth muscle tone (Moreland et al., 1985; Fernandez-Alfonso et al., 1992a,b).

ASM possesses a functional Na⁺-K⁺ pump that contributes to the maintenance of the resting membrane potential (Souhrada et al., 1981) and there is evidence that its activity can be regulated by bronchoactive agents. Vasoactive intestinal peptide stimulates the Na⁺-K⁺ pump in guinea pig tracheal ASM and this effect has been implicated in the bronchodilator response to this peptide (Morrison and Vanhoutte, 1996). A role for the Na⁺-K⁺ pump also has been proposed in relaxation of ASM in response to -adrenoceptor agonists, prostaglandin E₂, pituitary adenylate cyclase-activating peptide, and phorbol esters (Gunst and Stropp, 1988; Souhrada and Souhrada, 1989; Schramm and Grunstein, 1989; Kanemura et al., 1993; Tamaoki et al., 1994).

The aim of the present study was to examine the effect of 5-HT on the Na⁺-K⁺ pump of ASM. Na⁺-K⁺ pump activity was measured as ouabain-sensitive ⁸⁶Rb⁺ in cultured guinea pig tracheal ASM cells. The results indicate that 5-HT increases Na⁺-K⁺ pump activity in ASM through activation of 5-HT_2A receptors and that this effect may be secondary to stimulation of Na⁺ influx, possibly through the Na⁺-H⁺ exchanger. Stimulation of the Na⁺-K⁺ pump may represent a negative feedback mechanism that opposes contraction, and may, under certain circumstances, cause relaxation.

	Materials and Methods

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Cell Culture. Male Dunkin-Hartley guinea pigs (Covance, Denver, PA) with a body weight of 250 to 500 g were euthanized by an overdose of sodium pentobarbital (150 mg/kg i.p.). The trachea was excised, cleared of connective tissue, and cut longitudinally through the cartilage. The lumenal surface was gently rubbed with a sterile cotton-wool probe to remove the epithelium. The trachealis muscle was dissected away from the cartilage and minced with scalpel blades into ~1-mm² pieces. Trachealis pieces were placed in 150-mm diameter culture dishes containing culture medium and were maintained at 37°C in an incubator containing 5% CO₂ in humidified air. The culture medium consisted of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Within 1 week, cells migrated out of tissue explants and began to proliferate. On reaching confluence, cells were detached with 0.05% trypsin and 0.53 mM EDTA, centrifuged at 1500 rpm for 10 min, and resuspended in culture medium. Cells were either plated onto 24-well plates at a density of 2 to 4 × 10⁴ cells/well for ⁸⁶Rb⁺ uptake studies, or were subcultured onto 150-mm diameter dishes for up to five passages. Cells were identified immunohistochemically as smooth muscle cells by positive staining for smooth muscle-specific -actin (clone 1A4; Sigma Chemical Co., St. Louis, MO) and smooth muscle-specific myosin (clone hSM-V; Sigma Chemical Co.) with a labeled streptavidin-biotin-peroxidase kit (Zymed Laboratories, San Francisco, CA).

⁸⁶Rb⁺ Uptake. ⁸⁶Rb⁺ uptake, as a marker for K⁺ uptake, was measured in confluent ASM cells maintained at 37°C in a balanced salt solution (BSS) containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES and 10 mM glucose (pH 7.4). Following a 30 min equilibration period, cells were incubated for 10 min with BSS containing 0.5 µCi/ml ⁸⁶Rb⁺ and 1 nM to 0.1 mM 5-HT. Preliminary experiments indicated that uptake increased linearly with time for at least 10 min. Uptake was stopped by 6 washes with ice-cold BSS containing RbCl instead of KCl to displace ⁸⁶Rb⁺ from extracellular sites. Cells were solubilized in Lowry reagent and total cellular protein measured by the Lowry method (Sigma protein assay kit P5656). ⁸⁶Rb⁺ in the cellular digest was measured by liquid scintillation counting (3000-6000 dpm per sample counted for 10 min with a counting efficiency of 63%). The contribution of the Na⁺-K⁺ pump and the Na⁺-K⁺-Cl cotransporter to ⁸⁶Rb⁺ uptake was assessed with selective inhibitors to each pathway, ouabain for the Na⁺-K⁺ pump and bumetanide for the Na⁺-K⁺-Cl cotransporter. These two inhibitors have been previously shown to inhibit distinct pathways of ⁸⁶Rb⁺ uptake in guinea pig isolated tracheal smooth muscle (Rhoden and Douglas, 1995). Ouabain-sensitive uptake and bumetanide-sensitive uptake were estimated as the differences in uptake in the presence and absence of each inhibitor, added 5 min before ⁸⁶Rb⁺. The effect of various drug treatments on ⁸⁶Rb⁺ uptake was investigated by adding drugs or vehicle 5 min before ⁸⁶Rb⁺. The effect of reducing the extracellular concentration of Na⁺ on ⁸⁶Rb⁺ uptake was investigated by replacing NaCl in BSS with equimolar choline chloride. Due to the use of NaOH to adjust the pH of the medium, the final concentration of Na⁺ in the low Na⁺ BSS was 11 mM, whereas the concentration in the normal Na⁺ BSS was 146 mM.

Drugs. Bumetanide, dimethylamiloride (DMA) hydrochloride, 2,5-dimethoxy-4-iodoamphetamine (DOI) hydrochloride, 5-HT creatine sulfate, 5-(N-methyl-N-isobutyl)-amiloride (MIA), and ouabain were purchased from Sigma Chemical Co. 5-Carboxamidotryptamine (5-CT) maleate, 1-(3-chlorophenyl)piperazine (mCPP), 1-(3-chlorophenyl)biguanide (mCPB), ketanserin tartrate, -methyl-5-HT, NAN 190 hydrobromide, spiperone hydrochloride, and Y25130 hydrochloride were purchased from Tocris (Ballwin, MO). ⁸⁶RbCl was purchased from NEN (Boston, MA).

Data Analysis. Data are expressed as means ± S.E. of n experiments performed on cells cultured from different animals and/or passages. Within each experiment, duplicate or triplicate wells were used for each condition. EC₅₀ and pD₂ (log EC₅₀) values for agonists were determined by nonlinear regression curve fitting of concentration-response data fitted to the equation Y = Y_min + (Y_max  Y_min)/(1 + 10^{(logEC_{50log X)(Hill slope)}}), where Y is the response; Y_min and Y_max are the minimum and maximum responses, respectively; and X is the agonist concentration (GraphPad Prism, San Diego, CA). pK_B values for antagonists were calculated from the equation pK_B = log(DR  1)  log[antagonist] where DR is the ratio of the EC₅₀ values for 5-HT in the presence and absence of antagonist. Differences between groups were analyzed by ANOVA followed by Fisher's Protected Least Significant Difference (PLSD) post hoc test for multiple comparisons (StatView, SAS Institute Inc., Cary, NC). Statistical significance was assumed at a probability value <.05.

	Results

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Characterization of ⁸⁶Rb⁺ Uptake. ⁸⁶Rb⁺ uptake into cultured ASM cells increased linearly with time for at least 10 min and occurred at a rate of 20.0 ± 1.8 pmol/µg protein/min. Ouabain (10 nM-0.1 mM) induced a concentration-dependent inhibition of ⁸⁶Rb⁺ uptake with an EC₅₀ of 1.2 µM (pD₂ 5.89 ± 0.26, n = 5) and maximal inhibition of 41% occurring at 10 µM (Fig. 1A). Bumetanide (10 nM-0.1 mM) also induced a concentration-dependent inhibition of ⁸⁶Rb⁺ uptake with an EC₅₀ of 0.8 µM (pD₂ 6.10 ± 0.37, n = 8) and maximal inhibition of 37% occurring at 10 µM (Fig. 1A). Simultaneous addition of ouabain (10 µM) and bumetanide (10 µM) resulted in 85% inhibition of ⁸⁶Rb⁺ uptake, suggesting that the two inhibitors are acting on distinct and additive pathways (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effect of ouabain and bumetanide on 86Rb+ uptake into cultured guinea pig tracheal ASM cells. A, concentration-response curve for ouabain () and bumetanide (). B, effect of 10 µM ouabain (oua) and 10 µM bumetanide (bum) added simultaneously. Cells were incubated with ouabain and/or bumetanide for 5 min before exposure to 86Rb+ for 10 min. Basal uptake represents uptake in the absence of inhibitors. Points or bars represent mean ± S.E. of five to eight experiments (A) or eight experiments (B). *P < .001 versus basal, ANOVA with Fisher's PLSD test for multiple comparisons.

Effect of 5-HT on ⁸⁶Rb⁺ Uptake. 5-HT (1 nM-0.1 mM) induced a concentration-dependent increase in ⁸⁶Rb⁺ uptake that was abolished by 10 µM ouabain but was unaffected by 10 µM bumetanide (Fig. 2A), suggesting that it is mediated by the Na⁺-K⁺ pump and not the Na⁺-K⁺-Cl cotransporter. Furthermore, stimulation of ouabain-sensitive uptake by 5-HT was similar in the presence and absence of bumetanide, suggesting that the Na⁺-K⁺-Cl cotransporter does not modulate the effect of 5-HT on the Na⁺-K⁺ pump (Fig. 2B). 5-HT increased ouabain-sensitive ⁸⁶Rb⁺ uptake with an EC₅₀ of 30 nM (pD₂ 7.52 ± 0.18, n = 9) in the absence of bumetanide and 46 nM (pD₂ 7.34 ± 0.11, n = 9) in the presence of bumetanide. At a maximally effective concentration of 1 µM, 5-HT induced a 2-fold increase in ouabain-sensitive uptake. Ouabain- and bumetanide-insensitive uptake (i.e., uptake measured in the presence of both inhibitors) was not affected by 5-HT.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of 5-HT on 86Rb+ uptake into cultured guinea pig tracheal ASM cells. A, effect of 5-HT on total uptake () or uptake in the presence of 10 µM ouabain (), 10 µM bumetanide (), or both (). B, effect of 5-HT on ouabain-sensitive uptake in the absence () and presence () of 10 µM bumetanide. Cells were incubated with ouabain (10 µM) and/or bumetanide (10 µM) for 5 min before exposure to 86Rb+ and 5-HT for 10 min. Ouabain-sensitive uptake was calculated as the difference in uptake in the presence and absence of ouabain. Basal uptake represents uptake in the absence of 5-HT. Points represent means ± S.E. of nine experiments.

Effect of 5-HT Receptor Agonists and Antagonists. The receptor subtype mediating 5-HT-induced stimulation of the Na⁺-K⁺ pump was characterized with the use of selective 5-HT receptor agonists and antagonists. Ouabain-sensitive ⁸⁶Rb⁺ uptake, measured in the presence of 10 µM bumetanide, was stimulated by the 5-HT_2A/2C agonists -methyl-5-HT (pD₂ 7.26 ± 0.24, n = 9) and DOI (pD₂ 7.89 ± 0.21, n = 8) (Fig. 3). DOI was significantly more potent (P < .05) than -methyl-5-HT but not 5-HT. The potencies for -methyl-5-HT and 5-HT were not significantly different from each other. Ouabain-sensitive ⁸⁶Rb⁺ uptake was not affected by the 5-HT₁ agonist 5-CT, the 5-HT_1A/1B/2C agonist mCPP, or the 5-HT₃ agonist mCPB, all at concentrations of 10⁹ to 10⁵ M (n = 4).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of 5-HT2 agonists -methyl-5-HT () and DOI () on ouabain-sensitive 86Rb+ uptake. Cells were incubated with 86Rb+ and 5-HT agonists for 10 min. Ouabain-sensitive uptake was calculated as the difference in uptake in the presence and absence of 10 µM ouabain, added 5 min before 86Rb+. Basal uptake represents uptake in the absence of 5-HT agonists. Bumetanide (10 µM) was present throughout. Points represent means ± S.E. of nine (-methyl-5-HT) or eight (DOI) experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effect of the 5HT receptor antagonists ketanserin (A), spiperone (B), NAN 190 (C), and Y25130 (D) on 5-HT-stimulated ouabain-sensitive 86Rb+ uptake. Cells were incubated with antagonists (, 10 nM; , 100 nM; , 1 µM; , vehicle) for 5 min before exposure to 86Rb+ and 5-HT for 10 min. Ouabain-sensitive uptake was calculated as the difference in uptake in the presence and absence of 10 µM ouabain, added 5 min before 86Rb+. Basal uptake in the absence of 5-HT was subtracted from all data points. Bumetanide (10 µM) was present throughout. Points represent means ± S.E. of nine (ketanserin), seven (spiperone), six (NAN 190), and six (Y25130) experiments. Curves were fitted by nonlinear regression, with correlation coefficients of 0.94 to 0.99 for all curves.

Role of Na⁺ Influx and the Na⁺-H⁺ Exchanger. Intracellular Na⁺ activates the Na⁺-K⁺ pump, therefore changes in Na⁺-K⁺ pump activity may be secondary to changes in Na⁺ influx and hence intracellular Na⁺ concentration. To determine whether 5-HT-induced stimulation of ⁸⁶Rb⁺ uptake is dependent on Na⁺ influx, the concentration of Na⁺ in the extracellular medium was reduced from 146 mM to 11 mM (Fig. 5). Lowering extracellular Na⁺ caused a 62.4 ± 7.6% reduction in basal ouabain-sensitive ⁸⁶Rb⁺ uptake (P < .05, n = 5) presumably reflecting a decrease in intracellular Na⁺ concentration. In the presence of 11 mM Na⁺, 5-HT failed to induce a significant increase in ouabain-sensitive ⁸⁶Rb⁺ uptake (Fig. 5), suggesting that the ability of 5-HT to stimulate ⁸⁶Rb⁺ uptake is Na⁺-dependent.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of reducing extracellular Na+ concentration on 5-HT-induced stimulation of ouabain-sensitive 86Rb+ uptake. Cells were incubated with 86Rb+ and 1 µM 5-HT for 10 min in BSS containing normal (146 mM) or low (11 mM) Na+ concentration. Ouabain-sensitive uptake was calculated as the difference in uptake in the presence and absence of 10 µM ouabain, added 5 min before 86Rb+. Bumetanide (10 µM) was present throughout. Bars represent means ± S.E. of five experiments. *P < .001, 5-HT versus +5-HT; #P < .05 normal versus low Na+ BSS, ANOVA with Fisher's PLSD test for multiple comparisons.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Effect of Na+-H+ exchange inhibitors on 5-HT-stimulated ouabain-sensitive 86Rb+ uptake. Cells were incubated with DMA (A) or MIA (B) for 5 min before exposure to 86Rb+ and 1 µM 5-HT for 10 min. Ouabain-sensitive uptake was calculated as the difference in uptake in the presence and absence of 10 µM ouabain, added 5 min before 86Rb+. Bumetanide (10 µM) was present throughout. Bars represent means ± S.E. of five to eight experiments. **P < .001, *P < .01, 5HT versus +5-HT, ANOVA with Fisher's PLSD test for multiple comparisons.

	Discussion

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The results of this study suggest that 5-HT stimulates the Na⁺-K⁺ pump of ASM. 5-HT induced a 2-fold increase in ouabain-sensitive ⁸⁶Rb⁺ uptake without affecting bumetanide-sensitive uptake, suggesting that 5-HT selectively stimulates the Na⁺-K⁺ pump without affecting the Na⁺-K⁺-Cl^- cotransporter.

5-HT receptors are divided into seven classes (5-HT_1-7) comprising at least 14 subtypes based on their pharmacological profile, amino acid sequence, and signal transduction mechanisms (Hoyer et al., 1994). 5-HT-induced contraction of the guinea pig trachea is mediated by 5-HT_2A receptors (Baumgartner et al., 1990; Watts and Cohen, 1992, 1993) and the results of this study suggest that the same receptor subtype mediates stimulation of the Na⁺-K⁺ pump. DOI and -methyl-5-HT, agonists with high affinities for both the 5-HT_2A and 5-HT_2C subtypes, stimulated ouabain-sensitive ⁸⁶Rb⁺ uptake. In contrast, mCPP, an agonist on 5-HT_2C, 5-HT_1B, and 5-HT_1A receptors (in that order of potency) had no effect on uptake, suggesting that stimulation of uptake by DOI and -methyl-5-HT is probably due to activation of 5-HT_2A rather than 5-HT_2C receptors. Interestingly, DOI exhibited a bell-shaped concentration-response curve with high concentrations (10 µM) producing lesser stimulation than lower concentrations. This may be related to the rapid desensitization of 5-HT_2A receptors on guinea pig tracheal smooth muscle that is prominent during exposure to high agonist concentrations (Ben-Harari et al., 1991). Furthermore, DOI is a more potent agonist on 5HT_2A receptors than is -methyl-5-HT, therefore desensitization would be expected to be more evident for DOI.

Stimulation of ⁸⁶Rb⁺ uptake by 5-HT was antagonized by the 5-HT_2A antagonists ketanserin and spiperone, with pK_B values of 8.88 and 8.91, respectively. These values are in agreement with the known potencies of ketanserin and spiperone on 5-HT_2A receptors (Hoyer et al., 1994). Ketanserin appeared to cause not only a shift in the concentration-response curve to 5-HT but also a depression of the maximal response. Although ketanserin is known to be a competitive antagonist on 5-HT_2A receptors (Hoyer et al., 1994), it also has been reported to cause insurmountable inhibition of 5-HT-induced contraction of the guinea pig trachea (Lemoine and Kauman, 1986; Baumgartner et al., 1990; Watts and Cohen, 1992). This effect has been attributed to allosteric modulation of the 5-HT receptor by ketanserin, shifting the receptor from an active state to an inactive one and thereby decreasing the magnitude of the response (Lemoine and Kauman, 1986).

In vascular smooth muscle, 5-HT activates the Na⁺-K⁺ pump by two receptor subtypes coupled to distinct signal transduction pathways (Navran et al., 1991). 5-HT₁ receptors stimulate the Na⁺-K⁺ pump by a Ca²⁺-dependent activation of Na⁺ influx through the Na⁺-H⁺ exchanger, whereas 5-HT₂ receptors stimulate the pump directly via protein kinase C. In ASM, stimulation of ⁸⁶Rb⁺ uptake by 5-HT was prevented by reducing the extracellular concentration of Na⁺ and by pretreating cells with DMA and MIA, suggesting that stimulation of the pump is secondary to Na⁺ influx, possibly through the Na⁺-H⁺ exchanger. It should be pointed out that relatively high concentrations of DMA and MIA were required to inhibit uptake. The Na⁺-H⁺ exchanger exists in multiple isoforms (NHE1-6) that differ in their sensitivity to amiloride analogs, with NHE1 being the classical amiloride-sensitive isoform (Noel and Pouyssegur, 1995). DMA and MIA inhibit NHE1 in the low micromolar range, but are less potent on other isoforms. The low potency of DMA and MIA seen in this study may therefore reflect the presence of an isoform with a low amiloride sensitivity. Amiloride analogs, at concentrations >10 µM, also inhibit other Na⁺ channels, voltage-dependent Ca²⁺ channels, and several protein kinases (Kleyman and Cragoe, 1988). Despite the high concentrations needed, MIA was ~10-fold more potent than DMA in inhibiting 5-HT-induced ⁸⁶Rb⁺ uptake, and this is in agreement with their order of potency on the Na⁺-H⁺ exchanger (Kleyman and Cragoe, 1988). Furthermore, the two inhibitors had no effect on basal uptake, suggesting that DMA and MIA inhibit a pathway specifically activated by 5-HT.

What is the mechanism by which 5-HT_2A receptors increase Na⁺ influx through the Na⁺-H⁺ exchanger to mediate an increase in Na⁺-K⁺ pump activity? As with the Na⁺-K⁺ pump, the activity of the Na⁺-H⁺ exchanger is modulated by hormones, growth factors, and other extracellular signals (Noel and Pouyssegur, 1995). The Na⁺-H⁺ exchanger is activated through 1) phosphorylation by protein kinases A and C, 2) phosphorylation by a mitogen-activated protein kinase, 3) interaction with Ca²⁺-calmodulin at a specific site on the Na⁺-H⁺ exchanger, 4) interaction with cytoskeletal proteins, and 5) changes in intracellular pH. In ASM, 5-HT is known to activate inositol phosphate metabolism (Baumgartner et al., 1990; Yang et al., 1994b), Ca²⁺ mobilization (Yang et al., 1994a; Tolloczko et al., 1995), protein kinase C (Watts et al., 1994), and mitogen-activated protein kinase (Hershenson et al., 1995); therefore, a number of potential sites of interaction between 5-HT receptors and the Na⁺-H⁺ exchanger exist.

Stimulation of the Na⁺-K⁺ pump is associated with hyperpolarization (Souhrada and Souhrada, 1989). 5-HT induces contraction of the guinea pig trachea, in part, by stimulating Ca²⁺ influx through voltage-sensitive Ca²⁺ channels (Watts et al., 1994). Stimulation of the Na⁺-K⁺ pump would therefore be expected to reduce 5-HT-induced Ca²⁺ influx and oppose contraction. In ASM exposed to prolonged electrical stimulation, 5-HT induces rhythmic contractions, and the relaxant phase of these oscillations is abolished by ouabain (Kong and Stephens, 1990). Thus, 5-HT-induced stimulation of the Na⁺-K⁺ pump may represent a negative feedback mechanism that opposes contraction and may, under certain circumstances, give rise to relaxation. 5-HT also has been shown to relax precontracted guinea pig trachea (Bayol et al., 1985; Baumgartner et al., 1990; D'Agostino et al., 1996) and one may speculate whether this is due to stimulation of the Na⁺-K⁺ pump. However, the receptor mediating 5-HT-induced relaxation of guinea pig trachea is uncertain. One study has reported that relaxation is antagonized by ketanserin with an appropriate potency for inhibition at 5-HT_2A receptors (Baumgartner et al., 1990), but another study has reported 5-HT-induced relaxation in the presence of ketanserin (D'Agostino et al., 1996). Further studies are therefore needed to ascertain the role of the Na⁺-K⁺ pump in the relaxant response of ASM to 5-HT.

In conclusion, our results suggest that 5-HT stimulates the Na⁺-K⁺ pump of ASM through activation of 5-HT_2A receptors, and that stimulation is secondary to Na⁺ influx, possibly through the Na⁺-H⁺ exchanger. It is intriguing that a single receptor subtype is activating cellular pathways that have opposite effects on ASM tone, contraction through Ca²⁺ mobilization, and protein kinase C activation on the one hand, and inhibition of contraction through Na⁺-K⁺ pump activation on the other hand. It remains to be determined whether this is specific to 5-HT_2A receptors, or whether it is a general property of bronchoconstrictor receptors.