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Neurotrophins [e.g., brainderived 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.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]): sarcoplasmic reticulum (SR) [Ca.sup.2+] release and [Ca.sup.2+] influx (specifically store-operated [Ca.sup.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.sup.2+] indicator fura-2 AM. [[[Ca.sup.2+]].sub.i] responses to ACh, histamine, bradykinin, and caffeine and SOCE following SR [Ca.sup.2+] depletion were compared across cell groups. Force measurements were performed in human bronchial strips exposed to neurotrophins. Basal [[[Ca.sup.2+]].sub.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.sup.2+]].sub.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.sup.2+]].sub.i] and force regulation and suggest a potential role for neurotrophins in airway diseases. doi:10.1152/ajplung.00501.2004 brain-derived neurotrophic factor; neurotrophin 4; neurotrophin 3; sarcoplasmic reticulum; capacitative calcium entry

Sarcoplasmic reticulum (SR) [Ca.sup.2+] release and plasma membrane [Ca.sup.2+] influx are key to intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) regulation in airway smooth muscle (ASM). SR [Ca.sup.2+] depletion triggers influx via store-operated [Ca.sup.2+] channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated [Ca.sup.2+] influx in enzymatically dissociated porcine ASM cells. SR [Ca.sup.2+] was depleted by 1 [micro]M cyclopiazonic acid in 0 extracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.o]), nifedipine, and KCl (preventing [Ca.sup.2+] influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [[[Ca.sup.2+]].sub.o] and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 [micro]M isoproterenol or 100 [micro]M dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 [micro]M (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium1,2-diolate (DETA-NO nitric oxide donor) or 100 [micro]M 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated [Ca.sup.2+] influx was dependent on the extent of SR [Ca.sup.2+] depletion, sensitive to [Ni.sup.2+] and [La.sup.3+], but not inhibitors of voltage-gated influx channels, cAMP as well as cGMP potently inhibited [Ca.sup.2+] influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a [Ni.sup.2+]/[La.sup.3+]-sensitive [Ca.sup.2+] influx in ASM triggered by SR [Ca.sup.2+] depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., [beta]-agonists vs. nitric oxide). capacitative calcium entry; trachea; adenosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate; nitric oxide; isoproterenol

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 Ca2+ concentration ([Ca2+]i): sarcoplasmic reticulum (SR) Ca2+ release and Ca2+ influx (specifically store-operated Ca2+ 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 Ca2+ indicator fura-2 AM. [Ca2+]i responses to ACh, histamine, bradykinin, and caffeine and SOCE following SR Ca2+ depletion were compared across cell groups. Force measurements were performed in human bronchial strips exposed to neurotrophins. Basal [Ca2+]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 [Ca2+]i responses. ASM cells showed positive immunostaining for BDNF, NT3, NT4, trkB, and trkC (NT3 receptor). These novel data demonstrate that neurotrophins influence ASM [Ca2+]i and force regulation and suggest a potential role for neurotrophins in airway diseases. [ABSTRACT FROM AUTHOR]

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 μM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCI (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 μM isoproterenol or 100 μM dibutry 1 cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 μM (Z)-I-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 μM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels, cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., β-agonists vs. nitric oxide). [ABSTRACT FROM AUTHOR]