cyclic-gmp and tetramethylammonium

Tetraalkylammonium compounds and other organic cations were used to probe the structure of the internal and external mouths of the pore of cGMP-gated cation channels from rod and cone photoreceptors. Both rod and cone channels were blocked by tetramethyl- through tetrapentylammonium from the intracellular side in a voltage-dependent fashion at millimolar to micromolar concentrations. The dissociation constant at 0 mV (KD(O)) decreased monotonically with increasing carbon chain length from approximately 80 mM (TMA) to approximately 80 microM (TPeA), where the dissociation constant in rod channels is approximately 50% that of cone channels. N-Methyl-D-glucamine and the buffer Tris also blocked the cone channel in a voltage-dependent fashion at millimolar concentrations, but with lower affinity than similarly sized tetraalkylammonium blockers. Block by tetrahexylammonium (THxA) was voltage-independent, suggesting that the diameter of the intracellular mouth of these channels is less than the size of THxA but larger than TPeA. The location of the binding site for intracellular blockers was approximately 40% across the voltage-drop from the intracellular side. The addition of one carbon to each of the alkyl side chains increased the binding energy by approximately 4 kJ mol-1, consistent with hydrophobic interactions between the blocker and the pore. Cone, but not rod, channels were blocked by millimolar concentrations of extracellular TMA. The location of the extracellular binding site was approximately 13% of the voltage drop from the extracellular side. In cone channels, the two blocker binding sites flank the location of the cation binding site proposed previously.

Topics: Animals; Choline; Cyclic GMP; Ictaluridae; Ion Channel Gating; Ion Channels; Kinetics; Meglumine; Membrane Potentials; Quaternary Ammonium Compounds; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Tetraethylammonium; Tetraethylammonium Compounds

The dorsal integument of the medical leech Hirudo medicinalis exhibits a marked amiloride-sensitive Na+ absorption. With 20 mM Na+ in the apical solution, the transepithelial short-circuit current (Isc) was approximately 40% higher than with 115 mM Na+, whereas the transepithelial potential (VT) with 20 mM Na+ was -35.7 +/- 4.5 and -20.6 +/- 2.6 mV with 115 mM Na+. Amiloride (100 microM) inhibition at 20 mM apical Na+ was also significantly larger than with 115 mM Na+ in the solution. Benzamil (100 microM) showed additional inhibition after amiloride. Large transient overshooting currents occurred only when 115 mM Na+ was added after some minutes of Na(+)-free apical solution. Addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the serosal side in the presence of 115 mM apical Na+ nearly doubled Isc. This cAMP effect was reduced to only 20% in the presence of 20 mM Na+. Guanosine 3',5'-cyclic monophosphate (cGMP) slightly increased Isc, whereas ATP showed biphasic potency. Removal of calcium from the apical side resulted in a large stimulation of amiloride-sensitive Isc only in the presence of 115 mM Na+. When currents were activated with cAMP, a deprivation of Ca2+ modestly reduced the amiloride-sensitive Isc. The Na+ channel of leech integument was found highly selective for Na+ over other monovalent cations. The permeability ratio for Na+ over K+ was approximately 30:1; the selectivity relationship for the investigated cations was Na+ > Li+ > NH4+ > K+ approximately Cs+ approximately Rb+.

Topics: Amiloride; Animals; Calcium; Cyclic AMP; Cyclic GMP; Electrochemistry; Homeostasis; In Vitro Techniques; Ion Transport; Kinetics; Leeches; Quaternary Ammonium Compounds; Skin; Sodium; Sodium Channels

Nitric oxide (NO)-induced relaxation is associated with increased levels of cGMP in vascular smooth muscle cells. However, the mechanism by which cGMP causes relaxation is unknown. This study tested the hypothesis that activation of Ca-sensitive K (KCa) channels, mediated by a cGMP-dependent protein kinase, is responsible for the relaxation occurring in response to cGMP. In rat pulmonary artery rings, cGMP-dependent, but not cGMP-independent, relaxation was inhibited by tetraethylammonium, a classical K-channel blocker, and charybdotoxin, an inhibitor of KCa channels. Increasing extracellular K concentration also inhibited cGMP-dependent relaxation, without reducing vascular smooth muscle cGMP levels. In whole-cell patch-clamp experiments, NO and cGMP increased whole-cell K current by activating KCa channels. This effect was mimicked by intracellular administration of (Sp)-guanosine cyclic 3',5'-phosphorothioate, a preferential cGMP-dependent protein kinase activator. Okadaic acid, a phosphatase inhibitor, enhanced whole-cell K current, consistent with an important role for channel phosphorylation in the activation of NO-responsive KCa channels. Thus NO and cGMP relax vascular smooth muscle by a cGMP-dependent protein kinase-dependent activation of K channels. This suggests that the final common pathway shared by NO and the nitrovasodilators is cGMP-dependent K-channel activation.

Topics: Animals; Charybdotoxin; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Ethers, Cyclic; Male; Microelectrodes; Nitric Oxide; Okadaic Acid; Potassium Channels; Pulmonary Artery; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Scorpion Venoms; Thionucleotides; Vasodilation

Injection of cyclic AMP (cAMP) or cyclic GMP into identifiable neurones from several different gastropod species immediately depolarized the cell membranes in a dose-dependent manner. Doses were monitored photometrically and evidence is presented for depolarizing effects following nucleotide injections of as little as 30-35 mumol. The depolarizing effect was reversible and was demonstrated under voltage clamp to be primarily the result of a nucleotide-induced, transient increase in a membrane Na current, INa (cAMP). The charge-carrying species was identified by using ion-substituted salines, reversal potential in low-Na saline, and intracellular ion-sensitive electrode measurements. The current was resistant to tetrodotoxin, ouabain and amiloride. Substituting Trisma, tetramethylammonium or bis-tris propane for Na prevented the induced current, whereas Li substitution did not. Duration of the induced current was greatly prolonged in neurones bathed in the phosphodiesterase inhibitor isobutylmethylxanthine, or following injection of any of several cAMP analogues, indicating that the reversible nature of the current stems primarily from in situ hydrolysis of the injected dose and not current inactivation. Amplitude of the induced current either remained constant or decreased over the voltage range where it could be easily measured, i.e. -30 greater than Vm greater than -100 mV, reflecting a voltage as well as a chemical sensitivity of INa (cAMP).

Topics: 1-Methyl-3-isobutylxanthine; Action Potentials; Animals; Aplysia; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; In Vitro Techniques; Membrane Potentials; Mollusca; Neurons; Quaternary Ammonium Compounds; Ribonucleotides; Sodium; Tetrodotoxin

The mechanism of cholinergic stimulation of alanine and glutamine formation and release from skeletal muscle was studied using rat epitrochlaris preparations. The increased alanine and glutamine release produced by carbamylcholine (10(-6) M) was reproduced by tetramethylammonium (10(-6) M) but not by pilocarpine (10(-6) M) and was blocked by hexamethonium (10(-4) M) but not by atropine (10(-7) M). This increased alanine and glutamine release was not associated with altered muscle cAMP levels. However, carbamylcholine (10(-6) M) and tetramethylammonium (10(-6) M) did not increase levels of cGMP, 134% and 101%, respectively, and these increments in cGMP were blocked by hexamethonium but not by atropine. Carbamylcholine produced a concentration-dependent increase in cGMP levels. Methylisobutylxanthine and theophylline augmented the increased amino acid release and increased cGMP levels produced by carbamylcholine. Neither xanthine derivative alone altered alanine and glutamine release or cyclic nucleotide levels. Added cGMP increased amino acid release and the uptake of [U-14C]alanine and alpha-amino[14C]isobutyric acid. Carbamylcholine did not alter muscle phosphorylase a activity, glycogen levels, or basal adenylate cyclase activity. These data indicate that cholinergic stimulation of muscle alanine and glutamine formation and release involves a nicotinic cholinergic receptor and may be mediated by increased levels of cGMP, which in turn may result from a cholinergic stimulation of muscle guanylyl cyclase.

Topics: 1-Methyl-3-isobutylxanthine; Alanine; Animals; Atropine; Carbachol; Cyclic AMP; Cyclic GMP; Glutamine; Hexamethonium Compounds; Muscles; Phosphorylases; Pilocarpine; Quaternary Ammonium Compounds; Rats; Receptors, Cholinergic; Receptors, Nicotinic; Theophylline