ryanodine and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

Given that extracellular ATP is markedly elevated in inflammation and is known to modulate fibroblast function, we examined the effects of exogenously added ATP on Ca(2+)-handling and gene expression in human pulmonary fibroblasts. Cells were loaded with the Ca(2+)-indicator dye fluo-4 and studied using confocal fluorimetry. Standard RT-PCR was used to probe gene expression. ATP (10(-5)M) evoked recurring Ca(2+)-waves which were completely occluded by cyclopiazonic acid (depletes the internal Ca(2+)-store) or the phospholipase inhibitor U73122. Pretreatment with ryanodine (10(-5)M), however, had no effect on the ATP-evoked responses. Regarding the receptor through which ATP acted, we found the ATP-response to be mimicked by UTP or ADP but not by adenosine or alpha,beta-methylene-ATP, and to be blocked by the purinergic receptor blocker PPADS. The ATP-evoked response was greater and longer lasting within the nucleus than in the non-nuclear portion of the cytosol. RT-PCR showed that ATP also rapidly and dramatically increased gene expression of P2Y(4) receptors, the cytokine TGF-beta (an important modulator of wound repair) and two matrix proteins (collagen A1 and fibronectin) approximately 4-5 times above baseline: this increase was not significantly affected by ryanodine but was abolished by PPADS. We conclude that, in human pulmonary fibroblasts, ATP acts upon P2Y receptors to liberate internal Ca(2+) through ryanodine-insensitive channels, leading to a Ca(2+)-wave which courses throughout the cell and modulates gene expression.

Topics: Adenosine Triphosphate; Aniline Compounds; Caffeine; Calcium Signaling; Cell Nucleus; Cells, Cultured; Collagen; Estrenes; Fibroblasts; Fibronectins; Humans; Indoles; Lung; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrrolidinones; Ryanodine; Transforming Growth Factor beta; Type C Phospholipases; Xanthenes

1. Mechanisms underlying spontaneous rhythmical contractions have been studied in irideal arterioles of the rat using video microscopy and electrophysiology. 2. Rhythmical contractions (4 min-1) were more common during the second and third postnatal weeks and were always preceded by large, slow depolarizations (5-40 mV). 3. Spontaneous contractions were unaffected by tetrodotoxin (1 microM), neurotransmitter receptor antagonists, the sympathetic neurone blocker, guanethidine (5 microM) or sensory neurotoxin, capsaicin (1 microM). 4. Stimulation of sensory nerves inhibited spontaneous activity and this was not prevented by L-NAME (10 microm). 5. L-NAME (10 microm) caused an increase in frequency of spontaneous contractions, while forskolin (30 nM), in the presence of L-NAME, abolished spontaneous, but not nerve-mediated, contractions. 6. Spontaneous activity was not affected by felodipine (1 nM) or nifedipine (1 microM), but was abolished by cadmium chloride (1 microM) or superfusion with calcium-free solution. 7. Caffeine (1 mM), thapsigargin (2 microM) and cyclopiazonic acid (3 microM), but not ryanodine (3 microM), abolished spontaneous and nerve-mediated contractions. After preincubation in L-NAME (10 microM), cyclopiazonic acid abolished spontaneous contractions only. 8. Spontaneous depolarizations and contractions were abolished by 18alpha-glycyrrhetinic acid (20 microM). 9. Results suggest that spontaneous rhythmical contractions are myogenic and result from the cyclical release of calcium from intracellular stores, without a contribution from voltage-dependent calcium channels. Intercellular coupling through gap junctions appears to be essential for co-ordination of these events which could be modulated by nitric oxide and increases in cAMP. The possibility that different intracellular stores underly spontaneous and nerve-mediated contractions is discussed.

Topics: Adenosine Triphosphate; Age Factors; Animals; Arterioles; Cadmium Chloride; Caffeine; Calcium; Capsaicin; Central Nervous System Stimulants; Colforsin; Cyclic AMP; Dinucleoside Phosphates; Enzyme Inhibitors; Felodipine; Female; Gap Junctions; Iris; Male; Membrane Potentials; Muscle, Smooth, Vascular; Neuropeptide Y; NG-Nitroarginine Methyl Ester; Peptides, Cyclic; Periodicity; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Wistar; Ryanodine; Thapsigargin; Vasoconstriction; Vasodilator Agents