cyclic-gmp and tetraphenylphosphonium

1. The effects of human insulin and elevated D-glucose on L-arginine transport and synthesis of nitric oxide (NO) and prostacyclin (PGI2) have been investigated in human umbilical vein endothelial cells isolated from gestational diabetic pregnancies. 2. The increase in the Vmax for L-arginine transport (9.0 +/- 1.1) pmol (micrograms protein)-1 min-1) in diabetic endothelial cells cultured in 5 mM D-glucose was unaffected following 24 h exposure to 25 mM D-glucose. 3. Gestational diabetes-induced increases in basal intracellular cGMP and L-citrulline levels (inhibitable by L-NAME) and [Ca2+], were unaffected by elevated D-glucose. In contrast, PGI2 release was inhibited in diabetic cells exposed to either 5 or 25 mM D-glucose. 4. Elevated D-glucose attenuated histamine (10 microM, 5 min)-stimulated accumulation of cGMP and L-citrulline in endothelial cells isolated from gestational diabetic pregnancies. 5. The membrane hyperpolarization (-79 +/- 0.9 mV) sustained in diabetic endothelial cells in culture was insensitive to elevated D-glucose. 6. Elevated D-glucose abolished the stimulatory effect of human insulin (1 nM, 8 h) on L-[3H]leucine incorporation in diabetic endothelial cells cultured in 5 mM D-glucose. 7. Human insulin reduced the elevated rates of L-arginine transport and cGMP accumulation in diabetic cells cultured in 5 mM D-glucose but failed to reduce increased rates of transport or NO production in cells exposed to 25 mM D-glucose or cycloheximide. 8. Our findings demonstrate that hyperglycaemia impairs the actions of human insulin on umbilical vein endothelial cells isolated from gestational diabetic pregnancies. Changes in insulin sensitivity and/or its signalling cascade may be affected by hyperglycaemia associated with gestational diabetes, resulting in insulin resistance in endothelial cells derived from the fetal vasculature.

Topics: Amino Acids; Antimetabolites; Arginine; Calcium; Citrulline; Cyclic GMP; Deoxyglucose; Endothelium, Vascular; Epoprostenol; Female; Glucose; Humans; Insulin Resistance; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Pregnancy; Pregnancy in Diabetics; Umbilical Veins

Nitric oxide (NO) is known to potentiate neurotransmitter release in several types of neuronal cells. In the present study, the influence of NO on the membrane potential of isolated nerve endings (synaptosomes) from rat brain was studied. NO donors--sodium nitroprusside (SNP), S-nitroso-L-cysteine (CysNO), and hydroxylamine (HA)--induced synaptosome depolarization monitored by decreasing accumulation of 86Rb+ and the lipophilic potential-sensitive probe [3H]tetraphenylphosphonium. SNP reduced plasma membrane potential by 3-5 mV with half-maximal effect at approximately 10 microM. More potent NO donors, CysNO and HA, led to significant depolarization of the plasma membrane at 10-100 microM concentrations and also induced depolarization of mitochondria at concentrations above 1 mM. At 10 microM-10 mM concentrations, NO donors inhibited potassium channels; CysNO and HA also suppressed the activity of the sodium pump. NO-induced depolarization was not blocked by guanylate cyclase inhibitor methylene blue and the permeable cGMP analog dibutyryl-cGMP did not affect the membrane potential. The effects of NO donors were mimicked by SH-modifying reagents including 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). Non-permeable SH-reagent DTNB caused small depolarization resembling SNP action in its magnitude and kinetics. Significant decrease of potential in the presence of NEM, which permeates through the plasma membrane, was similar to that of CysNO and HA. The data suggest that in the presynaptic nerve endings, NO-induced depolarization of the plasma and mitochondrial membranes involves modification of protein SH-groups. The plasma membrane depolarization is due to the decreased potassium permeability and inhibition of the sodium pump.

Topics: Animals; Brain; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Kinetics; Male; Membrane Potentials; Methylene Blue; Nerve Endings; Nitric Oxide Donors; Onium Compounds; Organophosphorus Compounds; Potassium Channels; Rats; Rats, Wistar; Rubidium; Sodium Channel Blockers; Synaptosomes; Tissue Distribution

It has been proposed that both inositol trisphosphate and ryanodine receptors contribute to the Ca signal generated at fertilization of the sea urchin egg. Pharmacological studies indicate that cyclic adenosine diphosphate-ribose (cADPr) is the endogenous modulator of Ca release by the ryanodine-like receptor in eggs and that cADPR cyclase, the enzyme responsible for cADPR synthesis, can be stimulated by 3',5'-cyclic guanosine monophosphate (cGMP). Also, recent results show that the gaseous transmitter nitric oxide (NO) releases calcium in eggs via a mechanism linked to cGMP and cADPR production. Results reported here show that fertilization induces a rapid and transient increase in the intracellular concentration of cGMP. This increase occurs during the latent period, before the major increase in cytoplasmic free calcium (Cai), consistent with the hypothesis that cGMP production may play a key role in the Ca signal seen at fertilization.

Topics: Aminoquinolines; Animals; Calcium; Cyclic GMP; Cytosol; Enzyme Inhibitors; Female; Fertilization; Kinetics; Male; Membrane Potentials; Nitroprusside; Onium Compounds; Organophosphorus Compounds; Ovum; Sea Urchins; Sperm-Ovum Interactions; Spermatozoa

Topics: Animals; Carbachol; Cells, Cultured; Cyclic GMP; Kinetics; Membrane Potentials; Mice; Neoplasms, Experimental; Neuroblastoma; Onium Compounds; Organophosphorus Compounds; Potassium; Receptors, Cholinergic; Receptors, Muscarinic; Temperature