cyclic-gmp and calmidazolium

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3'-5' cyclic guanosine monophosphate (cGMP). Ca(2+)-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC(50) values of 11, 80 and 180 microM respectively. Here we sought to elucidate the underlying mechanism(s).. We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity.. Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca(2+) concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC(50)= 10 microM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V(max) and K(m), indicating that it acts in an uncompetitive manner.. The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.

Topics: Animals; Calcium; Calmodulin; Cerebellum; Cyclic GMP; Guanylate Cyclase; Imidazoles; Inhibitory Concentration 50; Nitric Oxide; Nitric Oxide Donors; Phenoxybenzamine; Rats; Rats, Wistar; Signal Transduction; Trifluoperazine

Natriuretic peptides (NPs) may work as neuromodulators through their associated receptors [NP receptors (NPRs)]. By immunocytochemistry, we showed that NPR-A and NPR-B were expressed abundantly on both ON-type and OFF-type bipolar cells (BCs) in rat retina, including the dendrites, somata, and axon terminals. Whole-cell recordings made from isolated ON-type BCs further showed that brain natriuretic peptide (BNP) suppressed GABAA receptor-, but not GABAC receptor-, mediated currents of the BCs, which was blocked by the NPR-A antagonist anantin. The NPR-C agonist c-ANF [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANF(4-23)-NH2] did not suppress GABAA currents. The BNP effect on GABAA currents was abolished with preincubation with the pGC-A/B antagonist HS-142-1 but mimicked by application of 8-bromoguanosine-3',5'-cyclomonophosphate. These results suggest that elevated levels of intracellular cGMP caused by activation of NPR-A may mediate the BNP effect. Internal infusion of the cGMP-dependent protein kinase G (PKG) inhibitor KT5823 essentially blocked the BNP-induced reduction of GABAA currents. Moreover, calcium imaging showed that BNP caused a significant elevation of intracellular calcium that could be caused by increased calcium release from intracellular stores by PKG. The BNP effect was blocked by the ryanodine receptor modulators caffeine, ryanodine, and ruthenium red but not by the IP3 receptor antagonists heparin and xestospongin-C. Furthermore, the BNP effect was abolished after application of the blocker of endoplasmic reticulum Ca2+-ATPase thapsigargin and greatly reduced by the calmodulin inhibitors W-7 and calmidazolium. We therefore conclude that the increased calcium release from ryanodine-sensitive calcium stores by BNP may be responsible for the BNP-caused GABAA response suppression in ON-type BCs through stimulating calmodulin.

Topics: Animals; Atrial Natriuretic Factor; Caffeine; Calcium; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Calmodulin; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; Guanylate Cyclase; Heparin; Imidazoles; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Membrane Potentials; Natriuretic Peptide, Brain; Oxazoles; Patch-Clamp Techniques; Peptide Fragments; Peptides, Cyclic; Polysaccharides; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor; Receptors, Cytoplasmic and Nuclear; Receptors, GABA; Receptors, GABA-A; Retinal Bipolar Cells; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin

Shear stress and tyrosine phosphatase inhibitors have been shown to activate the endothelial NO synthase (eNOS) in a Ca2+/calmodulin-independent manner. We report here that isometric contraction of rabbit aorta activates eNOS by a pharmacologically identical pathway. Endothelium-intact aortic rings were precontracted under isometric conditions up to 60% of the maximal phenylephrine-induced tone. The NO synthase inhibitor NGnitro-L-arginine (L-NA) and the soluble guanylyl cyclase inhibitor NS 2028 induced an additional contraction, the amplitude of which depended on the level of precontraction. The maximal production of NO by isometrically contracted aortic rings (as estimated by the increase in cGMP in detector smooth muscle cells in a superfusion bioassay) was observed during the initial phase of isometric contraction and was greater than that detected following the application of acetylcholine. The supplementary L-NA-induced increase in vascular tone was inhibited by the nonselective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A. Another tyrosine kinase inhibitor, genistein, the calmodulin antagonist calmidazolium, and the selective protein kinase C inhibitor, Ro 31-8220, had no effect. Coincident with the enhanced NO formation during isometric contraction was an increase in the tyrosine phosphorylation of endothelial proteins, which also correlated with the level of precontraction. Thus, isometric contraction activates eNOS via a Ca2+-independent, tyrosine kinase inhibitor-sensitive pathway and, like shear stress, seems to be an independent determinant of mechanically induced NO formation.

Topics: 1-Methyl-3-isobutylxanthine; Acetylcholine; Animals; Aorta, Thoracic; Benzoquinones; Calcium; Calmodulin; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Female; Genistein; Hydroquinones; Imidazoles; In Vitro Techniques; Indoles; Isometric Contraction; Lactams, Macrocyclic; Male; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Oxadiazoles; Oxazines; Quinones; Rabbits; Rifabutin; Staurosporine

Cyclic GMP (cGMP) formation induced by agonist stimulation of Ca2+/calmodulin-dependent nitric oxide (NO) synthase type I is known to occur in both granule cell and astrocyte cultures from rat cerebellum. Here we show that in these same cells cGMP is predominantly hydrolyzed by a Ca2+/calmodulin-dependent phosphodiesterase. At 10 microM cGMP, Ca2+ (25 microM) stimulated basal (Ca(2+)-independent) phosphodiesterase activity about 6 times in granular neurons and 15 times in astrocytes. The calmodulin antagonist calmidazolium blocked the Ca(2+)-dependent phosphodiesterase activity and exogenous calmodulin increased 3-4-fold the stimulatory potency of Ca2+ in both cell types (EC50 values 1.26 +/- 0.20 and 1.50 +/- 0.42 microM in the absence and 0.38 +/- 0.11 and 0.39 +/- 0.14 microM in the presence of 1 microM calmodulin, for neurons and astrocytes, respectively). In both cell types K(m) values for cGMP at 25 microM Ca2+ were similar (1.72 +/- 0.20 and 1.92 +/- 0.09 microM) and phosphodiesterase activities were inhibited by isozyme-selective phosphodiesterase inhibitors with potencies analogous to those described for Ca2+/calmodulin-phosphodiesterases or phosphodiesterase type 1 isoforms in other preparations. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) effectively blocked the Ca2+/calmodulin-phosphodiesterase activity in granule cell and astrocyte extracts (IC50 values at 1 microM cGMP: 31 +/- 10 microM and 46 +/- 6 microM, respectively), in contrast to the apparent inability of this compound to inhibit the Ca(2+)-dependent activity reported in whole brain extracts. These results demonstrate that comparable phosphodiesterase type 1 activities are found in the cytosols of cerebellar granule cells and astrocytes and suggest that these activities may play an important role in controlling cGMP levels in cells where the Ca(2+)-dependent NO synthase type I is stimulated.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Astrocytes; Calcium; Calmodulin; Cells, Cultured; Cerebellum; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 1; Enzyme Inhibitors; Granulocytes; Imidazoles; Lethal Dose 50; Neurons; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Rats; Rats, Sprague-Dawley

Regulation of guanosine 3',5'-cyclic monophosphate (cGMP) formation by calcium and calcium-binding proteins was studied at the levels of nitric oxide synthase (NOS) and guanylyl cyclase (GC) in dispersed pancreatic acini isolated from guinea pig. In intact cells, in the cytosol, and on diethylaminoethyl fractions from cytosolic proteins, GC activity was negatively regulated by Ca2+. An increase in Ca2+ concentration ([Ca2+]) from 25 to 950 nM suppressed cGMP formation by 85%. On the other hand, NOS was stimulated by agents increasing cytosolic [Ca2+] and inhibited by intracellular Ca2+ chelators. Thus Ca2+ regulates cGMP production in opposite directions by activating NOS and inhibiting GC. Calmodulin antagonists W-7, trifluoperazine, and R-24571 inhibited NOS, suggesting that the enzyme is regulated by calmodulin as in other cell types. Calmodulin antagonists appeared to inhibit GC. In particular, 200 microM W-7 completely abolished the cGMP rise evoked by the nitric oxide donor, nitroprusside. The effect was not reversed by addition of excess calmodulin. The findings suggest that the negative regulation of GC by Ca2+ is due to factors other than calmodulin but affected by calmodulin antagonists.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Amino Acid Oxidoreductases; Animals; Calcium; Calcium-Binding Proteins; Calmodulin; Cyclic GMP; Cytosol; Guanylate Cyclase; Guinea Pigs; Homeostasis; Imidazoles; In Vitro Techniques; Isoquinolines; Kinetics; Nitric Oxide Synthase; Pancreas; Piperazines; Protein Kinase C; Staurosporine; Sulfonamides; Trifluoperazine

Many antidepressants inhibit 5-hydroxytryptamine (5HT) transport resulting in increased 5HT levels in the synapse. However, physiological regulation of neurotransmitter uptake has not been demonstrated. We have examined the effect of receptor-activated second messengers on the 5HT transporter in rat basophilic leukemia cells (RBL 2H3). Here, we show that activation of an A3 adenosine receptor results in an increase of 5HT uptake in RBL cells, due to an increase in maximum velocity (Vmax). The A3 adenosine receptor-stimulated increase in transport is blocked by inhibitors of nitric oxide synthase and by a cGMP-dependent kinase inhibitor. In fact, compounds that generate nitric oxide (NO) and the cGMP analog 8-bromo-cGMP mimicked the effect of A3 receptor stimulation, suggesting that the elevation in transport occurs through the generation of the gaseous second messenger NO and a subsequent elevation in cGMP. Additionally, the 5HT transporter is differentially regulated by second messengers since direct activation of protein kinase C by phorbol esters decreases 5HT uptake by decreasing Vmax. Our results suggest that the changes in transport are due to a direct modification of the 5HT transporter, possibly by phosphorylation, which appears to alter the rate at which transport occurs. As the 5HT transporter in RBL cells is identical to that in neurons, our results suggest that analogous mechanisms may operate in the brain.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Amino Acid Oxidoreductases; Amino Acid Sequence; Aminoquinolines; Animals; Biological Transport; Carrier Proteins; Cell Membrane; Cyclic GMP; GTP-Binding Proteins; Imidazoles; In Vitro Techniques; Membrane Glycoproteins; Membrane Transport Proteins; Methylene Blue; Molecular Sequence Data; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Paroxetine; Protein Kinase C; Rats; Receptors, Purinergic P1; Second Messenger Systems; Serotonin; Serotonin Plasma Membrane Transport Proteins; Signal Transduction; Tumor Cells, Cultured

Relationships between cyclic nucleotides and cerebrovascular tone were investigated using closed cranial windows in anesthetized newborn pigs. Pial arteriolar diameter was monitored and cerebrospinal fluid (CSF) was collected from beneath the cranial window. Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) concentrations in CSF were 1,690 +/- 200 and 730 +/- 40 fmol/ml, respectively. Topically applied isozyme-selective and nonselective inhibitors [3-isobutyl-1-methylxanthine (IBMX), theophylline, Ro 201724, dipyridamole, zaprinast, calmidazolium, and W-7] of cyclic nucleotide phosphodiesterases dilated pial arterioles with concomitant increases in cAMP and/or cGMP levels in CSF. Topical application of dibutyryl-cAMP and dibutyryl-cGMP also resulted in pial arteriolar dilation. Ten-minute hypercapnia, which results in pial arteriolar dilation, increased cAMP to 5,240 +/- 900 and cGMP to 1,350 +/- 200 fmol/ml. IBMX and zaprinast potentiated the increases in cAMP and cGMP as well as the cerebrovascular dilation in response to hypercapnia. These data suggest that cyclic nucleotides contribute to regulation of cerebral vascular tone during control conditions. Furthermore, cAMP and/or cGMP appears to be involved in arterial vasodilation in response to hypercapnia in newborn pigs.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Animals, Newborn; Cerebrovascular Circulation; Cyclic AMP; Cyclic GMP; Hypercapnia; Imidazoles; Phosphodiesterase Inhibitors; Sulfonamides; Swine; Vasomotor System

Effects of the calmodulin inhibitor calmidazolium on stimulation of nitric oxide (NO) release were investigated in neuroblastoma N1E-115 cells. NO release was determined indirectly by measuring cyclic GMP formation. Instead of the expected decrease in NO generation based on the calmodulin dependence of neuronal NO synthase, calmidazoline paradoxically increased cyclic GMP formation. Maximal activation occurred at 3 min and the effects were concentration dependent. This calmidazolium-stimulated NO release was markedly blocked by hemoglobin and N-monomethyl-L-arginine.

Topics: Animals; Arginine; Calmodulin; Cyclic GMP; Dose-Response Relationship, Drug; Hemoglobins; Imidazoles; Kinetics; Mice; Neuroblastoma; Nitric Oxide; omega-N-Methylarginine; Tumor Cells, Cultured

1. Endothelin-3 (ET-3) at concentrations below those which caused contraction (30 nM) elicited endothelium-dependent relaxation followed by rebound contraction in rat isolated thoracic aorta. 2. Endothelin-1 also relaxed the rat aorta with a similar potency. 3. The nitric oxide (NO) synthase inhibitor, NG-nitro L-arginine, the radical scavenger, haemoglobin and the soluble guanylate cyclase inhibitor, methylene blue, each inhibited the ET-3-induced relaxation. 4. The calmodulin inhibitor, calmidazolium, considerably attenuated the relaxation caused by ET-3 without affecting that to nitroprusside. 5. Concentrations of ET-3 that were necessary to induce the relaxation also caused concentration-dependent elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels. 6. NG-nitro L-arginine, haemoglobin, methylene blue, calmidazolium and removal of the endothelium completely abolished ET-3-stimulated cyclic GMP production. 7. These results suggest that ET-3 triggers NO formation possibly via ETB receptors on the endothelium to activate soluble guanylate cyclase, which in turn stimulates cyclic GMP production and smooth muscle relaxation. The enzyme contributing to the NO formation may be of the calcium/calmodulin-dependent, constitutive type.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Arginine; Calmodulin; Cyclic GMP; Endothelins; Hemoglobins; Imidazoles; In Vitro Techniques; Male; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Rats; Rats, Wistar

The possibility that calmodulin inhibitors impair the constitutive but not the inducible nitric oxide synthase(s)-mediated inhibitions of tone was investigated in the rat aorta. The endothelium-dependent relaxations evoked by acetylcholine, ATP and the calcium ionophore A23187 (which are mediated by the constitutive nitric oxide synthase) were inhibited by calmodulin inhibitors [calmidazolium, W-7 and (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, hydrochloride, fendiline] and by an inhibitor of nitric oxide synthase, nitro L-arginine. Nitro L-arginine but not calmidazolium reduced the inhibitory influence of the endothelium on the concentration-contraction curves evoked by phenylephrine. Treatment of aortic rings without endothelium with interleukin-1 beta inhibited the contractions to phenylephrine by inducing nitric oxide synthase activity. Nitro L-arginine but not calmidazolium restored the contractility of the aortic rings. The relaxations evoked by a donor of nitric oxide, 3-morpholino-sydnonimine, were minimally affected by calmidazolium and nitro L-arginine. The basal tissue content in, and the production of, guanosine 3',5' cyclic monophosphate evoked by acetylcholine in rings with endothelium were inhibited by calmidazolium and nitro L-arginine. The production of cyclic GMP evoked by interleukin-1 beta in rings without endothelium was inhibited by nitro L-arginine but not by calmidazolium. These observations indicate that calmodulin inhibitors inhibit the constitutive but not the inducible nitric oxide synthase(s) in the rat aorta.

Topics: Amino Acid Oxidoreductases; Animals; Aorta; Arginine; Calmodulin; Cyclic GMP; Drug Interactions; Endothelium, Vascular; Fendiline; Imidazoles; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Inbred Strains; Vasodilator Agents

In this study we have investigated the role of oxygen delivery and of classic second messengers on erythropoietin production by the isolated perfused rat kidney. We found that the rat kidney was capable of de novo synthesis of erythropoietin. The erythropoietin production rate was inversely related to the oxygen pressure in the perfusate and increased from 0.17 to 1.85 U erythropoietin h-1 g kidney-1 when arterial PO2 was lowered from 500 mmHg to 30 mmHg. Addition of forskolin (10 microM) and 8-bromo-cGMP (100 microM) to the perfusate elicited significant effects on the renal vascular resistance, but had no significant effect on erythropoietin production. Hypoxia-induced erythropoietin formation, however, was blocked by calmidazolium (1 microM) and W-7 (10 microM), two structurally different putative calmodulin antagonists. Calmidazolium and W-7 had no effect on other functional parameters of the isolated perfused rat kidney such as flow rate, glomerular filtration rate or sodium reabsorption. Our findings suggest that the oxygen-sensing mechanism that controls renal erythropoietin production is primarily located in the kidney itself. A calcium/calmodulin-dependent cellular reaction could be involved in the signal transduction process.

Topics: Animals; Calmodulin; Colforsin; Cyclic GMP; Erythropoietin; Imidazoles; Kidney; Kinetics; Male; Oxygen; Rats; Second Messenger Systems; Sulfonamides; Vascular Resistance

The effects of calmidazolium, carbachol and membrane permeable derivatives of cGMP (dipalmitoyl cGMP and 8-Bromo cGMP) on the longitudinal internal resistivity (Ri) were studied in the rabbit atrial trabeculae by means of electrophysiological recording techniques and histological planimetry. Calmidazolium as well as carbachol decreased Ri whereas cGMP-derivatives enhanced this resistivity. The effect of calmidazolium suggested that calmodulin reduced the cell coupling under control conditions. Carbachol decreased the Ca-inward current, and probably it prevented the calmodulin activation. The action of the nucleotides showed that cGMP did not mediate the cholinergic effect on the cell coupling. The possible interaction between calmodulin and cGMP was discussed.

Topics: Animals; Carbachol; Cyclic GMP; Electric Stimulation; Heart; Imidazoles; In Vitro Techniques; Myocardium; Palmitates; Rabbits

The pharmacological and biochemical mechanisms of contractile responses to the protein kinase C (PKC) activator phorbol-12,13-diacetate (PDA) were investigated in canine basilar arteries. In the normal medium, PDA elicited a strong, dose-related, and slow-developing sustained contraction. Among the constrictors examined, including serotonin, prostaglandin F2 alpha, and endothelin, only PDA yielded contractions in a Ca2(+)-free medium. In both media, the PDA-induced contractions were virtually inhibited by either staurosporine, H-7, or quinacrine, while neither neurotransmitter blockades nor R24571 (calmidazolium) exerted significant effects. In addition, it was shown that 8-bromocyclic GMP, but not 8-bromocyclic AMP, markedly curtailed the PDA-induced contractions. Biochemical analysis, furthermore, showed that PDA induced increased phosphorylations of 27- and 96-kDa and proteins other than the myosin light chain (MLC) 20-kDa protein. Thus, the present results open up a novel mechanism of sustained cerebral artery contractions, where PKC activation rather than Ca2+/calmodulin/MLC system plays a key role that is regulated both by phospholipase A2 and by cyclic GMP.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Basilar Artery; Cyclic GMP; Dinoprost; Dogs; Endothelins; Enzyme Activation; Female; Imidazoles; Isoquinolines; Male; Muscle Contraction; Phorbol Esters; Phosphorylation; Piperazines; Protein Kinase C; Quinacrine; Serotonin; Staurosporine

Effects of 8-bromo-cGMP (8-Br-cGMP) and synthetic rat atriopeptin III (APIII) on sodium absorption by isolated chicken villus enterocytes and intact chicken ileal mucosa were determined. In isolated cells, both agents significantly decreased initial rates of influx of 22Na and caused a persistent decrease in intracellular pH (pHi); effects that are not additive to those caused by amiloride (10(-3) M). The ED50 for APIII was 0.3 nM. In intact mucosa, both 8-Br-cGMP (10(-4) M) and 5-(N-methyl-N-isobutyl)amiloride (MIBA) (10(-5) M) reduced JNams and JNa.net, their effects were not additive. APIII (10(-7) M) significantly increased cellular cGMP but not cAMP. Both 8-Br-cGMP (10(-4) M) and APIII (10(-7) M) stimulated a persistent increase in cytosolic calcium (Cai), which could be prevented by pretreating the cells with the cytosolic calcium buffering agent MAPTAM or with H-8, an inhibitor of cyclic nucleotide-dependent protein kinases. Furthermore, pretreatment of cells with H-8 or the calmodulin inhibitor, calmidazolium (CM), prevented the effects of 8-Br-cGMP and APIII on pHi. However, the pHi response to subsequent addition of the calcium-ionophore ionomycin was blocked only by CM and not by H-8. These data suggest that APIII and 8-Br-cGMP inhibit amiloride-sensitive Na/H exchange by increasing Cai, an event requiring activation of cGMP-dependent protein kinase.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amiloride; Animals; Atrial Natriuretic Factor; Biological Transport; Calcium; Carrier Proteins; Chickens; Cyclic GMP; Hydrogen-Ion Concentration; Ileum; Imidazoles; In Vitro Techniques; Intestinal Absorption; Intestinal Mucosa; Isoquinolines; Sodium; Sodium-Hydrogen Exchangers

1. The effects of three phosphodiesterase inhibitors (papaverine, isobutyl methyl xanthine (IBMX) and SKF 94120) were examined on tension responses and cyclic nucleotide content (both cyclic AMP and cyclic GMP) of normal and Triton X-100 skinned isolated trachealis of the guinea-pig. 2. The three inhibitors were approximately equipotent in eliciting concentration-dependent relaxation of histamine-induced contractions of the trachealis. 3. Papaverine-induced relaxation was associated with concentration-related increases in the levels of both cyclic nucleotides. 4. IBMX at low concentrations (1 mumol l-1) produced significant relaxation (36%) of histamine-contracted trachealis without changing cyclic nucleotide levels. At a ten fold higher concentration IBMX-induced relaxation (95%) was associated with a selective increase in tissue cyclic GMP levels. Only at the highest concentration tested (100 mumol l-1) did IBMX increase cyclic AMP levels significantly. 5. SKF 94120 (1 mumol l-1) elicited a 23% relaxation of the contracted trachealis without altering the tissue content of either cyclic nucleotide. At the two higher concentrations tested (10 and 100 mumol l-1), SKF 94120-induced relaxation was accompanied by a selective increase in the levels of cyclic AMP. 6. In the skinned trachealis Ca2+ (10 and 20 mumol l-1)-induced contractions were significantly inhibited by the calmodulin antagonist calmidazolium (10 mumol l-1) and by cyclic AMP (10 mumol l-1), the catalytic subunit of cyclic AMP-dependent protein kinase (0.1 mumol l-1) and cyclic GMP (10 mumol l-1). 7. Papaverine (100 mumol l-1) significantly inhibited (31 +/- 6%) the Ca2+-induced contractions of the skinned trachealis. Both IBMX and SKF 94120 were without effect. It is concluded that cyclic nucleotide-dependent mechanisms have an inhibitory action on the biochemical processes that lead to contraction of the guinea-pig trachealis. The results suggest that a functional sarcoplasmic reticular and/or plasma membrane is essential for the expression of IBMXand SKF 94120-induced relaxation. This is not the case for papaverine. The results also highlight the fact that significant relaxant responses of airway smooth muscle can be produced by phosphodiesterase- inhibiting drugs without concomitant elevations in tissue cyclic nucleotide content.

Topics: 1-Methyl-3-isobutylxanthine; Airway Resistance; Animals; Cyclic AMP; Cyclic GMP; Guinea Pigs; Histamine; Imidazoles; In Vitro Techniques; Male; Papaverine; Phosphodiesterase Inhibitors; Protein Kinases; Pyrazines