No abstract

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 Ca2+ signaling, Ca2+ 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 Ca2+ oscillations in airway SMCs that resulted from Ca2+ influx through L-type voltage-gated Ca2+ channels and the subsequent release of Ca2+ from intracellular stores through ryanodine receptors. Phorbol ester stimulation of lung slices in which SMC intracellular Ca2+ concentration ([Ca2+]i) was “clamped” at a high concentration induced strong airway contraction, indicating that PKC mediated sensitization of the contractile response to [Ca2+]i. This Ca2+ 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 Ca2+ 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 Ca2+ oscillations and strong Ca2+ 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 Ca2+ entry and induce airway hyperresponsiveness.

No abstract

Transthyretin (TTR) is an attractive candidate for use in phylogenetic analysis because it is a short, single-copy nuclear gene with regions that are highly conserved across evolutionarily-divergent organisms from Xenopus laevis to Homo sapiens. To explore its utility as a phylogenetic marker, the complete intron one region (789–805 bp) was sequenced in 22 crocodylian species. Detailed analyses of intron 1 resolved the three expected lineages, Alligatorids, Crocodylids, and Gavialids, and offered additional evidence for the utility of synapomorphic indels in elucidating higher-level phylogenetic relationships. When used in conjunction with other genetic and morphological data sets, intron 1 should be a valuable tool in the investigation of other closely-related taxa.

No abstract

Nucleotide sequence comparisons have identified a gene product in the genome database of African clawed frogs (Xenopus laevis) as a probable member of the solute carrier family of membrane transporters. To confirm its identity as a putative iodide transporter, we examined the function of this sequence after heterologous expression in mammalian cells. A green monkey kidney cell line transfected with the Xenopus nucleotide sequence had significantly greater (125)I uptake than sham-transfected control cells. The uptake in carrier-transfected cells was significantly inhibited in the presence of perchlorate, a competitive inhibitor of mammalian Na(+)/iodide symporter. Tissue distributions of the sequence were also consistent with a role in iodide uptake. The mRNA encoding the carrier was found to be expressed in the thyroid gland, stomach, and kidney of tadpoles from X. laevis, as well as the bullfrog Rana catesbeiana. The ovaries of adult X. laevis also were found to express the carrier. Phylogenetic analysis suggested that the putative X. laevis iodide transporter is orthologous to vertebrate Na(+)-dependent iodide symporters. We conclude that the amphibian sequence encodes a protein that is indeed a functional Na(+)/iodide symporter in X. laevis, as well as R. catesbeiana.

Protein kinase C (PKC) is an important regulator of smooth muscle cell (SMC) contraction; however the mechanisms and the PKC isoforms responsible for this regulation in small intrapulmonary airways are unknown. We have investigated the effects of pharmacological stimulation of PKC on Ca2+ signalling, Ca2+ sensitivity, and contraction in airway SMCs using mouse lung slices and phase‐contrast and confocal microscopy. Activation of PKC with phorbol 12‐myristate 13‐acetate (PMA, 1–10 μM) induced low frequency Ca2+ oscillations and transient airway contractions (twitching). However, a strong and sustained airway contraction was induced by PMA in Ca2+‐permeabilized lung slices with caffeine and ryanodine. This contraction was blocked by the PKC inhibitor GF‐109203X (1 μM), but not by the Rho kinase inhibitor Y‐27632 (10 μM). Furthermore, PMA treatment increased KCl‐induced airway contraction. Using RT‐PCR and specific oligonucleotide primers, the presence of mRNAs encoding PKCβ, PKCγ, and PKCζ were identified in the airways. In conclusion, our results suggest that activation of specific PKC isoforms induce airway contraction by stimulating Ca2+ oscillations and Ca2+ sensitivity in SMCs.