Ca2+ regulation of vascular smooth muscle

Regulation of intracellular free Ca2+ concentrations in vascular smooth muscle is accomplished mainly by Ca2+ channels and ATP-dependent Ca2+ pumps in the plasmalemma and sarcoplasmic reticulum (SR). Ca2+ entry through the plasmalemma is apparently mediated by four different pathways: leak; receptor-operated Ca2+ channels; potential sensitive Ca2+ channels; and stretch-activated channels. The agonist releasable intracellular Ca2+ store appears to be identical with the SR. Evidence for the involvement of Ca2+-induced Ca2+ release and inositol-1,4,5-trisphosphate in the release of SR Ca2+ is discussed. Smooth muscle contractions induced by certain agonists may be further enhanced by inhibition of Ca2+ uptake by the SR and of active Ca2+ extrusion across the plasmalemma. At the moment it is not clear from a consideration of the Ca2+ regulatory mechanisms present in vascular smooth muscle how dietary Ca2+ affects vascular tone. The increased Ca2+ permeation through smooth muscle cell membranes of resistance arteries taken from spontaneously hypertensive rats may be relevant to this problem.     

Ca2+ regulation in vascular smooth muscle.

Regulation of aorta smooth muscle contraction by Ca ion requires the collaboration of the 80,000 dalton factor and tropomyosin. A method for preparing pure actin from aorta smooth muscle is described.     

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

Ca2+-sensitivity and cGMP-independent effects of NO in vascular smooth muscle.

The effects of NO on Ca2+-sensitivity of vascular smooth muscle (VSM) myofilaments have been the focus of this study. Simultaneous measurements of [Ca2+]i and force were carried out in rat tail artery segments. NO, 10(-7) M, evoked a transient decrease in [Ca2+]i accompanied by sustained relaxation (45.3+/-6.3 vs. 69.45+/-7.2%, P<0.05, respectively) of VSM precontracted with K+ (70 mM), suggesting a decrease in Ca2+-sensitivity of VSM. This decrease in Ca2+-sensitivity was completely abolished by preincubation of VSM with ODQ (10(-6) M) (63.9+/-7.8% for [Ca2+]i vs. 20.5+/-8.4% for relaxation, P<0.05). Ca2+-presensitization of VSM myofilaments with PE (10(-6) M) decreased the efficacy of NO to relax VSM (44.25+/-6.9% vs. 69.45+/-7.2%, P<0.05), but increased its ability to lower [Ca2+]i (70.5+/-6.8% vs. 45.3+/-6.3%, P<0.05). Application of DTT (10(-3) M) together with ODQ (10(-6) M) to subtract possible cGMP-independent effects revealed the total suppression of both the relaxant responses and [Ca2+]i of VSM under high-K+ preactivation of VSM. The data indicate that NO not only relaxes VSM and lowers [Ca2+]i in K+-preactivated VSM, but also decreases Ca2+-sensitivity of VSM myofilaments and these effects are strongly cGMP-dependent. In PE-induced contractions of VSM, NO relaxed VSM of rat tail artery and lowered [Ca2+]i, but failed to reverse Ca2+-presensitized myofilaments. We suggest that alternative cGMP-independent effects of NO are primarily manifested via activation of K+-channels and inhibition of Ca2+ current rather than to affect relaxation. An importance of reduced SH-groups within VSM myoplasm for both relaxation and [Ca2+]i disposal evoked by NO is evident whatever Ca2+-mobilization pathways are involved.     

A Ca2(+)-activated, Mg2(+)-dependent ATPase with high affinities for both Ca2+ and Mg2+ in vascular smooth muscle microsomes: comparison with plasma membrane Ca2(+)-pump ATPase

A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mg2+-Ca2+ interaction in contractility of vascular smooth muscle: Mg2+ versus organic calcium channel blockers on myogenic tone and agonist-induced responsiveness of blood vessels

Contractility of all types of invertebrate and vertebrate muscle is dependent upon the actions and interactions of two divalent cations, viz, calcium (Ca2+) and magnesium (Mg2+) ions. The data presented and reviewed herein contrast the actions of several organic Ca2+ channel blockers with the natural, physiologic (inorganic) Ca2+ antagonist, Mg2+, on microvascular and macrovascular smooth muscles. Both direct in vivo studies on microscopic arteriolar and venular smooth muscles and in vitro studies on different types of blood vessels are presented. It is clear from the studies done so far that of all Ca2+ antagonists examined, only Mg2+ has the capability to inhibit myogenic, basal, and hormonal-induced vascular tone in all types of vascular smooth muscle. Data obtained with verapamil, nimopidine, nitrendipine, and nisoldipine on the microvasculature are suggestive of the probability that a heterogeneity of Ca2+ channels, and of Ca2+ binding sites, exists in different microvascular smooth muscles; although some appear to be voltage operated and others, receptor operated, they are probably heterogeneous in composition from one vascular region to another. Mg2+ appears to act on voltage-, receptor-, and leak-operated membrane channels in vascular smooth muscle. The organic Ca2+ channel blockers do not have this uniform capability; they demonstrate a selectivity when compared with Mg2+. Mg2+ appears to be a special kind of Ca2+ channel antagonist in vascular smooth muscle. At vascular membranes it can (i) block Ca2+ entry and exit, (ii) lower peripheral and cerebral vascular resistance, (iii) relieve cerebral, coronary, and peripheral vasospasm, and (iv) lower arterial blood pressure. At micromolar concentrations (i.e., 10-100 microM). Mg2+ can cause significant vasodilatation of intact arterioles and venules in all regional vasculatures so far examined. Although Mg2+ is three to five orders of magnitude less potent than the organic Ca2+ channel blockers, it possesses unique and potentially useful Ca2+ antagonistic properties.     

Modulation of plasma membrane Ca2+ pump by membrane potential in cultured vascular smooth muscle cells

We examined the effect of membrane potential (Em) on the activity of the plasma membrane Ca2+ pump in cultured rat aortic smooth muscle cells (VSMCs). Inside-negative K+ diffusion potential higher or lower than the resting Em (-46 mV) was artificially imposed on VSMCs with various concentrations of extracellular K+ (K+o) and 1 microM valinomycin. We found that the recovery phase of the intracellular Ca2+ transient elicited with 1 microM ionomycin was accelerated by depolarizing Em, whereas it was retarded by hyperpolarizing Em. The rate of extracellular Na+ (Na+o)-independent 45Ca2+ efflux from VSMCs stimulated with 1 microM ionomycin increased almost linearly with a change in Em from -98 to -3 mV. This effect of Em was abolished by extracellularly added LaCl3 or a combination of high pH (pH 8.8) and high Mg2+ (20 mM), conditions that presumably inhibit the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., & Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Intracellular contents of Na+ and K+ and intracellular pH, on the other hand, were not influenced by the change in Em under the conditions used. These results indicate that alteration in Em can modulate the intracellular Ca2+ concentration in intact VSMCs by changing the rate of Ca2+ extrusion by the plasma membrane Ca2+ pump. The data strongly suggest that the plasma membrane Ca2+ pump in VSMCs is electrogenic.     

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.     

T-type Ca2+ channels in vascular smooth muscle: multiple functions

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

Mg2+ restores membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate movements

Cellular ornithine biosynthesis could be expected to play a significant role in putrescine formation and hence in growth. Two enzymes are involved in ornithine biosynthesis: arginase and transamidinase. These enzyme activities were studied in two human melanoma cell lines differing in their K_m of diamine oxidase for putrescine and in their tumorigenicity in nude mice. Arginase activity accounts for the majority of ornithine formed in the highly tumorigenic cell line, while the majority of ornithine is derived from transamidinase action in the poorly tumorigenic cell line, with concomitant formation of methyl guanidine, a potent inhibitor of diamine oxidase.     

Effect of the membrane potential on the Mg2+,ATP-dependent transport of Ca2+ across smooth muscle sarcolemma

The effect of the membrane potential (K(+)-valinomycin system) on the Mg2+, ATP-dependent transport of Ca2+ in inside-out vesicles of myometrium sarcolemma has been studied. The membrane potential was identified by using a cyanine potential-sensitive probe, diS-C3-(5). In the presence of valinomycin (5.10(-8) M) the inside-out directed K+ gradient (delta psi = -86 mV, with a negative charge inside) stimulated the initial rate of the energy-dependent accumulation of Ca2+ transfer whereas the oppositely directed K+ gradient (delta psi = +72 mV, with a positive charge inside) had no effect on this process. The K+ gradient was formed by isotonic substitution of K+ in intra- or extravesicular space for choline +. At the same time, in the absence of K+ gradient the Mg2+, ATP-dependent accumulation of Ca2+ in membrane vesicles did not depend on the chemical nature of the cations (K+ or choline+) used for isotonicity. The decrease of delta psi from 0 to -86 mV affects the initial rate of Ca2+ accumulation but not the maximal content of the accumulated cation. Preliminary dissipation of the membrane potential (delta psi = -86 mV) in Mg2(+)-free isotonic (with respect of K+ and choline+) media containing ATP and Ca2+ resulted in the inhibition of Mg2+, ATP-dependent Ca2+ transport induced by subsequent addition of Mg2+. These results indicate that the negative (intravesicular) electrical potential activates the Ca-pump of smooth muscle sarcolemma. This activation is based on the increase in the turnover number of the Ca2+ transporting system but not on its affinity for the transfer substrate. The use of the absolute reaction rates theory made it possible to establish that the Ca-pump effectuates the transport of a single positive charge in inside-out vesicles of smooth muscle plasma membranes, i.e., the energy-dependent transport of Ca2+ occurs either as a symport (with an anion (Cl-) or an antiport with a monovalent cation (K+) or a proton. It is assumed that the potential dependence of the Ca-pump in the smooth muscle plasma membrane plays a role in the realization of effects of mediators and physiologically active substances that are manifested as stimulation of the contractile response and depolarization of the sarcolemma. In is quite probable that the delta psi-dependent Ca-pump is also responsible for the maintenance of intracellular homeostasis of monovalent cations (K+, H+, Cl-) in smooth muscle tissues.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle: effects of Sr2+ and Cd2+ on Ca2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATPase

The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase.     

Neuropeptide Y-induced intracellular Ca2+ increases in vascular smooth muscle cells

The effect of neuropeptide Y (NPY) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured smooth muscle cells from porcine aorta (PASMC) and compared with the effect of bradykinin (BK) and angiotensin II (ATII) on [Ca2+]i. All peptides induced dose-dependent and transient rises in [Ca2+]i which were not blocked by extracellular EGTA, but the NPY response was different from the others' as follows. First, the [Ca2+]i rise induced by NPY was not as rapid as that induced by BK or ATII. Second, pertussis toxin abolished the [Ca2+]i rise induced by NPY, but not by BK or ATII. Third, following initial treatment with BK, PASMC were able to respond to NPY, but not to ATII. Finally, BK and ATII, but not NPY, significantly increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation. Although NPY attenuated forskolin-induced accumulation of cyclic AMP, forskolin- and 3-isobutyl-1-methyl-xanthine-induced alterations in intracellular cyclic AMP did not affect the NPY-induced [Ca2+]i rise. These results suggest that NPY increases [Ca2+]i by a pertussis toxin-sensitive GTP binding protein-involved mechanism which is not mediated by the intracellular messengers such as Ins(1,4,5)P3 and cyclic AMP.     

The effects of EGTA on vascular smooth muscle contractility in calcium-free medium

The effect of EGTA, commonly present in Ca2+-free physiological saline solution, on the contractile responses induced by Ca2+ and phenylephrine was studied in dog mesenteric arteries and aortas of rats and rabbits. EGTA substantially enhanced the contractile responses of these vascular strips or rings to added Ca2+ after a prolonged preincubation period in the Ca2+-free medium. The maximal level of the enhanced contractile responses was independent of EGTA concentration, but the rate of the maximal responses was faster at higher EGTA concentration, presumably as a result of faster removal of intracellular Ca2+. Such a Ca2+-induced response was sensitive to the Ca2+ antagonist, nifedipine. EGTA present at low concentrations (50 and 400 microM) in Ca2+-free medium also inhibited the phenylephrine-induced contractile response more prominently for the longer preincubation periods of the vascular tissues in Ca2+-free medium. Our results suggest that EGTA, even when added at low concentrations to the vascular smooth muscle for a sufficiently long period in Ca2+-free medium, may cause destabilization of the cell membranes leading to increased permeability to subsequently added Ca2+. EGTA may also remove the superficially bound Ca2+ and subsequently reduce the intracellular Ca2+ pool via extraction of the intracellular Ca2+ at the cell membrane surfaces.     

Ca2+ microdomains in smooth muscle

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Comparison of the effects of smooth and skeletal muscle actins on smooth muscle actomyosin Mg2+-ATPase

Actin has been purified from smooth muscle (chicken gizzard) by two different procedures and its activation of smooth muscle myosin Mg2+-ATPase activity compared with that achieved with rabbit skeletal muscle actin. The procedure of Pardee and Spudich (Methods Enzymol. (1982) 85, 164-181) for the purification of rabbit skeletal muscle actin is readily applicable to the isolation of chicken gizzard actin, enabling large quantities to be purified in two days. Smooth muscle actin could be successfully stored as F-actin at -80 degrees C and survived freezing and thawing at least twice. Smooth muscle actin activated myosin Mg2+-ATPase to a higher level than its skeletal muscle counterpart (77.9 nmol Pi/min/mg myosin vs 48.1 nmol Pi/min/mg myosin).     

Neurotrophin effects on intracellular Ca2+ and force in airway smooth muscle

Neurotrophins [e.g., brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT4)], known to affect neuronal structure and function, are expressed in nonneuronal tissues including the airway. However, their function is unclear. We examined the effect of acute vs. prolonged neurotrophin exposure on regulation of airway smooth muscle (ASM) intracellular Ca(2+) concentration ([Ca(2+)](i)): sarcoplasmic reticulum (SR) Ca(2+) release and Ca(2+) influx (specifically store-operated Ca(2+) entry, SOCE). Human ASM cells were incubated for 30 min in medium (control) or 1 or 10 nM BDNF, NT3, or NT4 (acute exposure) or overnight in 1 nM BDNF, NT3, or NT4 (prolonged exposure) and imaged after loading with the Ca(2+) indicator fura-2 AM. [Ca(2+)](i) responses to ACh, histamine, bradykinin, and caffeine and SOCE following SR Ca(2+) depletion were compared across cell groups. Force measurements were performed in human bronchial strips exposed to neurotrophins. Basal [Ca(2+)](i), peak responses to all agonists, SOCE, and force responses to ACh and histamine were all significantly enhanced by both acute and prolonged BDNF exposure (smaller effect of NT4) but decreased by NT3. Inhibition of the BDNF/NT4 receptor trkB by K252a prevented enhancement of [Ca(2+)](i) responses. ASM cells showed positive immunostaining for BDNF, NT3, NT4, trkB, and trkC (NT3 receptor). These novel data demonstrate that neurotrophins influence ASM [Ca(2+)](i) and force regulation and suggest a potential role for neurotrophins in airway diseases.