8-bromocyclic-gmp and alpha-beta-methyleneadenosine-5--triphosphate

The development of nerve-induced activation, receptor properties and cellular signalling was examined by comparing the urinary bladder of new-born (0-2 days) and adult mice. Tissue strips were isolated and the effects of different neuromuscular agents on force were investigated during electrical field stimulation. The nerve-induced contractions of the urinary bladders from new-born mice were less influenced by desensitisation with alpha, beta-methylene ATP and more sensitive to scopolamine compared with those of the adult bladder. There were no differences in alpha, beta-methylene ATP or ATP responsiveness between adult and new-born tissue, showing that the lower purinergic component of the nerve-induced responses in the new-born mice was due to properties of the transmitter release rather than to a change in receptor function. Dose-response curves for carbachol revealed a lower peak response in new-born bladders compared with adults. The phasic component of the cholinergic contractions was pronounced and initiated at low carbachol concentrations in the new-born tissue. The carbachol contractions of both new-born and adult urinary bladder tissue were inhibited by the Rho kinase inhibitor Y27632 and by the protein kinase G activator 8-Br-cGMP. However, the sustained phase of carbachol contraction was significantly less sensitive to Y27632 and 8-Br-cGMP in new-born tissue. These results suggest that the receptor mediated calcium sensitisation mechanism is less prominent in new-born compared with adult mice and that the contractions of new-born bladders are less influenced by the nitric oxide (NO)-cGMP inhibitory pathway.

Topics: Adenosine Triphosphate; Age Factors; Amides; Animals; Animals, Newborn; Carbachol; Cholinergic Agonists; Cholinergic Fibers; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electric Stimulation; Female; In Vitro Techniques; Male; Mice; Mice, Inbred Strains; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxants, Central; Muscle, Smooth; Nifedipine; Pyridines; Receptors, Muscarinic; Scopolamine; Tetrodotoxin; Urinary Bladder; Vasodilator Agents

We investigated the role of K+ channels and intracellular Ca2+ stores in the relaxations induced by the NO donor 3-morpholinosydnonimine (SIN-1) and 8-bromo-cGMP (8-BrcGMP), 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP), and alpha, beta-methylene-ATP in isolated segments of rat ileum. The inhibitory responses to SIN-1 and the cGMP analogs were not influenced by the K+ blockers apamin, charybdotoxin, iberiotoxin, or glibenclamide, whereas relaxations induced by alpha,beta-methylene-ATP were abolished by apamin and tetraethylammonium. The NO-donor SIN-1 and the cGMP analogs were able to inhibit contractions induced by activation of L-type Ca2+ channels (BAY-K-8644), by carbachol (CCh), and by cyclopiazonic acid (CPA), a blocker of sarcoplasmic Ca2+-ATPase. However, the inhibition of the combined CPA and CCh response was reduced and the dose-response curve of SIN-1 shifted to the right. Intracellular Ca2+ stores were emptied by incubation in Ca2+-free buffer and repetitive stimulation with CCh or BAY-K-8644. After restoration of extracellular Ca2+, the inhibitory effect of SIN-1 and pCPT-cGMP was only attenuated, whereas in the additional presence of CPA, the inhibitory effect of SIN-1 was blocked and the effect of 8-BrcGMP reduced. Thus depleting intracellular Ca2+ stores attenuated the effect of SIN-1 and 8-BrcGMP, suggesting an involvement of functional Ca2+ stores.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Animals; Apamin; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Carbachol; Charybdotoxin; Cyclic GMP; Egtazic Acid; Glyburide; Ileum; In Vitro Techniques; Indoles; Kinetics; Male; Molsidomine; Muscle Contraction; Muscle, Smooth; Peptides; Potassium Channels; Rats; Rats, Wistar; Tetraethylammonium; Tetrodotoxin; Thionucleotides