cyclic-gmp and indo-1

In sepsis, lipopolysaccharide (LPS) depresses cardiac function by inducing production of nitric oxide (NO) and its second messenger cGMP. LPS also stimulates ANG II production. We hypothesized that ANG II modulates the cardiac response to LPS. Adult rabbit cardiac myocytes incubated with LPS (10 ng/ml) had increased cardiac cGMP after 6 h (but not within 1 h) [527 +/- 43 vs. 316 +/- 27 (SE) fmol/mg protein in controls, n = 16 each group, P < 0.05]. This was associated with depressed cell shortening with no alterations in Ca2+ transients (indo 1 fluorescence), indicating a decreased myofilament responsiveness to Ca2+. ANG II (100 nM) alone had no effect. However, ANG II with LPS produced higher cGMP levels (1,025 +/- 113 fmol/mg protein, n = 16, P < 0.05 vs. LPS alone), more severe contractile depression, impaired Ca2+ handling, and decreased mitochondrial activity (MTS assay). We conclude that ANG II and LPS have synergistic effects on the activation of NO-cGMP pathways to induce dose-dependent impairments in excitation-contraction coupling in cardiac myocytes.

Topics: Angiotensin II; Animals; Antihypertensive Agents; Calcium; Cells, Cultured; Chelating Agents; Cyclic GMP; Enzyme Inhibitors; Female; Heart Ventricles; Imidazoles; Indoles; Lipopolysaccharides; Losartan; Male; Mitochondria; Muscle Fibers, Skeletal; Myocardial Contraction; Nitric Oxide; omega-N-Methylarginine; Pyridines; Rabbits; Receptors, Angiotensin; Sepsis; Ventricular Function

1. Intracellular calcium concentration ([Ca2+]i) was measured in mouse whole islets of Langerhans using the calcium-sensitive fluorescent dye Indo-1. 2. Application of physiological concentrations of 17beta-oestradiol in the presence of a stimulatory glucose concentration (8 mM) potentiated the [Ca2+]i signal in 83 % of islets tested. Potentiation was manifested as either an increase in the frequency or duration of [Ca2+]i oscillations. 3. The effects caused by 17beta-oestradiol were mimicked by the cyclic nucleotide analogues 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) and 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP). 4. Direct measurements of both cyclic nucleotides demonstrated that nanomolar concentrations of 17beta-oestradiol in the presence of 8 mM glucose increased cGMP levels, yet cAMP levels were unchanged. The increment in cGMP was similar to that induced by 11 mM glucose. 5. Patch-clamp recording in intact cells showed that 8-Br-cGMP reproduced the inhibitory action of 17beta-oestradiol on ATP-sensitive K+ (KATP) channel activity. This was not a membrane-bound effect since it could not be observed in excised patches. 6. The action of 17beta-oestradiol on KATP channel activity was not modified by the specific inhibitor of soluble guanylate cyclase (sGC) LY 83583. This result indicates a likely involvement of a membrane guanylate cyclase (mGC). 7. The rapid decrease in KATP channel activity elicited by 17beta-oestradiol was greatly reduced using Rp-8-pCPT-cGMPS, a specific blocker of cGMP-dependent protein kinase (PKG). Conversely, Rp-cAMPS, which inhibits cAMP-dependent protein kinase (PKA), had little effect. 8. The results presented here indicate that rapid, non-genomic effects of 17beta-oestradiol after interaction with its binding site at the plasma membrane of pancreatic beta-cells is a cGMP-dependent phosphorylation process.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Animals; Calcium; Calcium Signaling; Cell Membrane; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Enzyme Inhibitors; Estradiol; Fluorescent Dyes; Indoles; Islets of Langerhans; Male; Mice; Mice, Inbred Strains; Patch-Clamp Techniques; Potassium Channels; Thionucleotides

Previous studies in isolated cardiac myocytes suggest that impaired relaxation during reoxygenation after brief hypoxia results from abnormal Ca(2+)-myofilament interaction. Recent studies indicate that guanosine 3',5'-cyclic monophosphate (cGMP)-elevating interventions selectively enhance myocardial relaxation. We investigated the effect of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on posthypoxic relaxation in single rat myocytes, with simultaneous measurement of contraction and intracellular Ca2+ (indo 1 fluorescence). In control myocytes (n = 11), reoxygenation after 10 min of hypoxia markedly prolonged time to peak shortening (+36.5 +/- 4.2%) and half-relaxation time (+75.7 +/- 11.3% cf. normoxic values; both P < 0.001) and reduced diastolic length but did not change cytosolic Ca2+. Under normoxic conditions, 50 microM 8-BrcGMP slightly reduced time to peak shortening and half-relaxation time and increased diastolic length but did not alter cytosolic Ca2+. In the presence of 8-BrcGMP, there was no posthypoxic delay in twitch relaxation nor was there a decrease in diastolic length (half-relaxation time -5.8 +/- 3.3% cf. normoxic values; P < 0.05 cf. control group; n = 11). Cytosolic Ca2+ remained unaltered. Thus, 8-BrcGMP fully prevents impaired posthypoxic relaxation in isolated cardiac myocytes, probably by altering Ca(2+)-myofilament interaction.

Topics: Animals; Calcium; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Fluorescent Dyes; Heart; Indoles; Myocardial Contraction; Myocardial Ischemia; Myocardium; Oxygen; Rats; Rats, Wistar; Time Factors

Using the probe indo-1 in a microspectrofluorimetric study, it was demonstrated that the locust antidiuretic neurohormone, neuroparsin, enhanced cytosolic free Ca2+ concentrations, measured as percentages of the ratio F405/F480 (R), in epithelial cells of the African locust rectum. 5-hydroxytryptamine, whose antidiuretic effect was previously established, enhanced R in longitudinal muscular cells, and was able to increase R slightly in epithelial cells. The possibility of reciprocal Ca2+ movements between muscular and epithelial cells is discussed. Both of the neuronal molecules, which act via distinct transduction pathways (phosphoinositide turnover for neuroparsin, and Ca(2+)-dependent adenylate cyclase for 5-hydroxytryptamine), stimulated an increase in R by causing Ca2+ entry through dihydropyridine-sensitive Ca2+ channels. Cyclic nucleotides (cAMP and cGMP) lowered R in epithelial cells, the cGMP effect being interpreted as a feedback control on phosphoinositide turnover and resulting in the ability to re-establish cAMP production to levels incompatible with high PLC activity. In longitudinal muscular cells, the increase in R due to cAMP suggests the involvement of 5-hydroxytryptamine in stimulation of adenylate cyclase activity. Furthermore, results enabled the localization in epithelial cells of the transduction pathways mediating the actions of another antidiuretic factor extracted from the glandular lobes of the locust corpora cardiaca.

Topics: Animals; Calcium; Cyclic AMP; Cyclic GMP; Grasshoppers; Indoles; Insect Hormones; Male; Neurotransmitter Agents; Serotonin

We have studied the contractility of liver sinusoidal stellate (Ito) cells stimulated with endothelin 1, nitric-oxide donors and eicosanoids. Contraction and relaxation of stellate cells were detected by the use of a silicone-rubber method that revealed the traction forces exerted by these cells. Endothelin 1 was a strong elicitor for stellate-cell contraction. 78, 55, 59 and 56% of stellate cells were contracted 2.5, 5, 10 and 20 min, respectively, after exposure to 10 nM endothelin 1. The effect of endothelin 1 was dose dependent and still detectable at an endothelin 1 concentration of 100 pM. Concomitantly, an endothelin-dependent formation of inositol phosphates was apparent; values of InsP, InsP2, and InsP3 were 881 +/- 99%, 1965 +/- 368%, and 791 +/- 120% of control, respectively, 20 min after addition of 10 nM endothelin 1. In addition, endothelin 1 caused a transient increase of [Ca2+]i in stellate cells from a basal value of 121 +/- 9 nM to maximal 1015 +/- 86 nM. These endothelin-1 effects were much stronger than those of the thromboxane-A2 analogue U46619 and of prostaglandin F2 alpha. In contrast, Iloprost, prostaglandin E2, and sodium nitroprusside promoted stellate-cell relaxation; for example, 82, 83 and 71% of stellate cells relaxed 5, 10, and 20 min, respectively, after addition of 500 microM sodium nitroprusside to contacted cells. Prostaglandin E2 and Iloprost led to elevation of cAMP levels in stellate cells from a basal value of 9.2 +/- 0.8 pmol/well to 55.1 +/- 8.0 and 122.2 +/- 12.2 pmol/well 10 min after addition of prostaglandin E2 (5 microM) and Iloprost (5 microM), respectively, in the presence of 3-isobutyl-1-methylxanthine (0.5 mM). However, sodium nitroprusside was a trigger for cGMP accumulation. Intracellular cGMP increased from a basal value of 0.9 +/- 0.07 pmol/well to 13.4 +/- 6.7 pmol/well 10 min after addition of 500 microM sodium nitroprusside into the medium. It is interesting that Iloprost and sodium nitroprusside also induced the disappearance of actin stress fibers in contracted cells; F-actin stress fibers became less numerous and de-aggregated; more than 90% of stellate cells were void of stress fibers after 10 microM Iloprost treatment for 30 min. Thus, endothelin 1, eicosanoids and sodium nitroprusside are able to modulate the contractility of stellate cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blotting, Western; Bucladesine; Calcium; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cytoskeleton; Cytosol; Dibutyryl Cyclic GMP; Dinoprost; Endothelins; Enzyme Activation; Fluorescent Dyes; GTP-Binding Proteins; Iloprost; Indoles; Inositol Phosphates; Kinetics; Liver; Male; Nitric Oxide; Nitroprusside; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Wistar; Time Factors; Type C Phospholipases; Vasoconstrictor Agents

Receptors for atrial natriuretic peptide (ANP) have been demonstrated in renal mesangial cells as well as other cell types in the glomerulus. The biochemical basis for the effects of ANP on glomerular hemodynamics remains undefined. Using cultured rat glomerular mesangial cells, we demonstrated a concentration-dependent stimulation of cGMP production in intact cells, and of guanylate cyclase in membranes. Despite the presence of a guanylate cyclase response, ANP had no inhibitory effect on basal inositol trisphosphate production nor on basal cytosolic calcium. Arginine vasopressin stimulated IP3 production, caused a rise in cytosolic calcium as measured using the calcium-sensitive fluorescent probe Indo-1, and caused mesangial cell contraction. ANP caused a slight but significant enhancement of vasopressin-stimulated IP3 production, but had no effect on the cytosolic calcium response nor on the contractile response. 8-Bromo-cGMP likewise had no effect on the generation of the calcium signal. These results indicate that the effects of ANP on glomerular hemodynamics are not mediated by an alteration in the generation of the calcium signal in mesangial cells. In contrast, addition of calcium inhibited ANP stimulated guanylate cyclase activity.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Calcium; Cells, Cultured; Cyclic GMP; Cytosol; Fluorescent Dyes; Guanylate Cyclase; Indoles; Inositol Phosphates; Kidney Glomerulus; Rats; Rats, Inbred Strains; Vasopressins