Ca2+ oscillations, Ca2+ sensitization, and contraction activated by protein kinase C in small airway smooth muscle.

Protein kinase C (PKC) has been implicated in the regulation of smooth muscle cell (SMC) contraction and may contribute to airway hyperresponsiveness. Here, we combined optical and biochemical analyses of mouse lung slices to determine the effects of PKC activation on Ca(2+) signaling, Ca(2+) sensitivity, protein phosphorylation, and contraction in SMCs of small intrapulmonary airways. We found that 10 µM phorbol-12-myristate-13-acetate or 1 µM phorbol 12,13-dibutyrate induced repetitive, unsynchronized, and transient contractions of the SMCs lining the airway lumen. These contractions were associated with low frequency Ca(2+) oscillations in airway SMCs that resulted from Ca(2+) influx through L-type voltage-gated Ca(2+) channels and the subsequent release of Ca(2+) from intracellular stores through ryanodine receptors. Phorbol ester stimulation of lung slices in which SMC intracellular Ca(2+) concentration ([Ca(2+)](i)) was "clamped" at a high concentration induced strong airway contraction, indicating that PKC mediated sensitization of the contractile response to [Ca(2+)](i). This Ca(2+) sensitization was accompanied by phosphorylation of both the PKC-potentiated PP1 inhibitory protein of 17 kD (CPI-17) and the regulatory myosin light chain. Thrombin, like the phorbol esters, induced a strong Ca(2+) sensitization that was inhibited by the PKC inhibitor GF-109203X and also potentiated airway contraction to membrane depolarization with KCl. In conclusion, we suggest that PKC activation in small airways leads to both the generation of Ca(2+) oscillations and strong Ca(2+) sensitization; agents associated with airway inflammation, such as thrombin, may activate this pathway to sensitize airway smooth muscle to agonists that cause membrane depolarization and Ca(2+) entry and induce airway hyperresponsiveness.

Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle.

Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K(+) channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders.

Tumor necrosis factor-α (TNF-α) augments AMPA-induced Purkinje neuron toxicity.

It is well recognized that exposure of neurons to excessive levels of the excitatory neurotransmitter glutamate, termed glutamate excitotoxicity, contributes to the damage and degeneration seen in many acute and chronic neurological diseases. However, it is becoming increasingly evident that inflammation also can play a role in certain neurodegenerative diseases and inflammatory mediators, such as tumor necrosis factor-α (TNF-α), may directly interact with excitotoxic processes. In a postnatal rat cerebellar slice model, we found that TNF-α exacerbated AMPA-induced excitotoxicity in Purkinje neurons in a dose-dependent manner beyond the toxicity caused by AMPA alone. It also was shown that combinations of TNF-α and AMPA increased the mean intracellular activity of calpains, calcium-activated cysteine proteases that are known to contribute to cell death in Purkinje neurons. Additionally, these combinations augmented colbalt influx, a marker for calcium entry that selectively occurs through calcium permeable AMPA receptors only. Pharmacologic blockade of calcium permeable AMPA receptors with a specific antagonist, 1-naphthyl acetyl spermine (NASPM), reversed the apparent increase in AMPA receptor calcium permeability caused by TNF-α as measured by cobalt influx; caused a reduction in the Purkinje neuron calpain activity; and reversed the enhanced neurodegeneration induced by the combination of TNF-α and AMPA. From these studies we concluded that TNF-α augmented AMPA-induced toxicity in Purkinje neurons by increasing intracellular calcium flux through calcium permeable AMPA receptors, and this increase in calcium was directly involved in enhanced activation of calpains and a greater percentage of Purkinje neuron loss.