Phenytoin inhibition of cyclic guanosine 3':5'-monophosphate (cGMP) accumulation in neuroblastoma cells by calcium channel blockade

Phenytoin (diphenylhydantoin) inhibits the calcium-dependent increases in guanosine 3':5'-monophosphate (cGMP) produced by high potassium depolarization and by muscarinic receptor activation in N1E-115 neuroblastoma cells. The inhibition of the cGMP response to depolarization is half-maximal at 40 microM, similar to the plasma concentration associated with an optimal therapeutic response. The cGMP increase produced by the cationophore A23187 is insensitive to phenytoin blockade, indicating that the enzymatic machinery responsible for calcium-stimulated cGMP accumulation is not affected. The calcium concentration-response curve for the cGMP response to high potassium showed that phenytoin acted primarily to reduce the maximal response. The corresponding curve for the cGMP response to acetylcholine showed apparent competitive inhibition by phenytoin whereas the acetycholine concentration-response curve showed noncompetitive inhibition by phenytoin. The results suggest that phenytoin inhibits cGMP responses by blocking calcium influx. The ability to block the depolarization-induced cGMP response is shared by other anticonvulsants which are effective against generalized tonic-clonic and cortical focal seizures but not by those effective against absence seizures.     

Impairment of vascular and platelet levels of nitric oxide and cyclic guanosine-3',5'-monophosphate in cyclosporin A-induced hypertensive rats

The therapeutic use of the immunosuppressive agent cyclosporin A (CsA) is associated with arterial hypertension and increased risk of thromboembolism. Impaired endothelium-mediated relaxation is one of the main hypotheses explaining the CsA-induced vascular hyper-reactivity. Since nitric oxide (NO) modulates both vascular and platelet activity, we studied the effects of CsA on the levels of arterial and platelet NO as well as 3',5'-cyclic guanosine monophosphate (cGMP) levels which are influenced by NO. An animal model of CsA-induced hypertension was used. Wistar rats were treated with a clinically relevant, oral dose of 5 mg/kg CsA, daily, for 4 weeks. CsA increased both systolic and diastolic blood pressures compared to non-treated rats (P < 0.01). Nitrite, a NO metabolite, decreased in the entire aorta wall (30%, P < 0.05) and in the aorta wall without the endothelial layer (70 %, P < 0.05) in CsA-treated vs. control groups. cGMP content was also decreased in the CsA-treated group (67%, P < 0.01) vs. control. Taken together, these results suggest a defect on the endothelial NO generation, acceleration of breakdown and/or consumption of NO, as well as marked alterations directly on cGMP metabolism. Conversely, platelet nitrite and cGMP content significantly increased in the CsA-treated rats, which was also observed in in vitro studies of platelet nitrite release following CsA treatment. This suggests a platelet self-regulation mechanism against CsA-induced platelet hyper-reactivity, which, in turn, could compensate vascular impairment.     

Long-lasting cyclic guanosine-3',5'-monophosphate accumulation in the medium of cultured smooth muscle cells from atherosclerotic rabbit aortas in response to exogenous or endogenous nitric oxide

Although atherosclerosis causes a marked inhibition of the endothelium-dependent vasorelaxation it also leads to expression of inducible nitric oxide synthase (iNOS), accompanied by an increase in cyclic GMP content, in the arterial wall. The aim of our present study as to evaluate the influence of atherosclerosis on the soluble guanylyl cyclase pathway in viable cultured smooth muscle cells (SMC) from rabbit atherosclerotic rabbit aortas (atherosclerotic SMC) and from control rabbit aortas (control SMC). In response to 100 microM sodium nitroprusside (SNP), the intracellular production of cyclic GMP was similar in both types of cells, reaching a maximum after 5 min of incubation. In the culture medium, SNP evokes an increased cyclic GMP concentration lasting 6 h in control SMC and 24 h in atherosclerotic SMC. Interleukin-1beta (100 IU/mL), which induces iNOS in SMC from both control and atherosclerotic aortas causes accumulation of cyclic GMIP in the extracellular medium between 3 and 6 h for control SMC and between 3 and 24 h with atherosclerotic SMC. These results demonstrate a long-lasting egression of cyclic GMP in the extracellular medium of cultured SMC from rabbit aortas in response to endogenous or exogenous NO. Since this egression of cyclic GMP lasts longer in atherosclerotic than in control SMC, we suggest that atherosclerosis dysregulates the long-term soluble guanylyl cyclase response to NO in SMC.     

Interaction of nitric oxide and cyclic guanosine 3',5'-monophosphate in erythropoietin production.

The present study was designed to investigate whether in vivo and in vitro erythropoietin (EPO) production is modulated by nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP). Serum levels of EPO in ex-hypoxic polycythemic mice were significantly increased after injections of 200 micrograms/kg sodium nitroprusside for 4 d. One injection of NG-nitro-L-arginine methyl ester (L-NAME) produced a significant dose-related decrease in serum levels of EPO in ex-hypoxic polycythemic mice in response to hypoxia. When EPO producing Hep3B cells were incubated in 1% O2 for 30 min, cGMP levels in the Hep3B cells were significantly elevated, compared with cells incubated in 20% O2. The elevation of cGMP by hypoxia was inhibited by L-NAME (100 microM). Sodium nitroprusside (10 and 100 microM) and NO (2 microM) also significantly increased cGMP levels in Hep3B cells. L-NAME, LY 83583 (6-Anilino-5,8-quinolinedione, a soluble guanylate cyclase inhibitor), and Rp-8-Bromo-cGMPS (Rp-8-Bromo-guanosine 3',5'-cyclic monophosphothioate, a cGMP-dependent protein kinase inhibitor) significantly inhibited the hypoxia-induced increase in medium levels of EPO in Hep3B cells. 8-Bromo-cGMPS produced a dose-dependent decrease in EPO messenger RNA levels in Hep3B cells in response to hypoxia. 8-Bromo-cGMP (10(-3) M) produced significant increases in medium levels of EPO in Hep3B cell cultures incubated under normoxic conditions, which was enhanced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (0.2 mM). These results suggest that NO and cGMP may interact in modulating hypoxic stimulation of EPO production.     

Urinary adenosine cyclic 3',5'-monophosphate in idiopathic calcium stone-formers: response to an oral calcium load

Idiopathic calcium stone-formers with hypercalciuria during fasting have significantly lower urinary cyclic AMP levels (nmol/dl of glomerular filtrate) than fasting normocalciuric stone-formers. Female subjects, including both normal subjects and idiopathic calcium stone-formers, have higher urinary cyclic AMP levels than their male counterparts, and this difference is significant when urinary cyclic AMP is expressed in the units mumol/g of creatinine. Expressing urinary cyclic AMP in nmol/dl of glomerular filtrate reduces this difference but does not abolish it. Thus, in comparing urinary cyclic AMP levels in various subgroups of the calcium stone-formers and in normal subjects, both sex differences and the units of urinary cyclic AMP expression must be taken into consideration. The magnitude of the change in urinary cyclic AMP in response to an oral calcium load appears to depend on the antecedent urinary cyclic AMP excretion rate, whereby those individuals (either normal subjects or calcium stone-formers) having the highest urinary cyclic AMP levels demonstrate the greatest fall in urinary cyclic AMP after a calcium load.     

Endothelin (ET)-3 stimulates cyclic guanosine 3',5'-monophosphate production via ETB receptor by producing nitric oxide in isolated rat glomerulus, and in cultured rat mesangial cells.

We investigated the effects of endothelins on receptor-mediated cyclic nucleotide metabolism in rat glomerulus, inner medullary collecting duct (IMCD), and also in cultured rat glomerular mesangial cells. Endothelin (ET)-3 dose-dependently stimulated cGMP accumulation in glomerulus, which was higher than that of ET-1 or ET-2. ETB receptor agonist IRL 1620 produced cGMP in a dose-dependent manner, mimicking the effect of ET-3. ETA receptor antagonist BQ123-Na did not inhibit ET-3- or IRL 1620-stimulated cGMP generation. NG-monomethyl-L-arginine (L-NMMA) significantly inhibited ET-3- or IRL 1620-induced cGMP production, suggesting that ET-3- or IRL 1620-stimulated cGMP generation was mediated through nitric oxide (NO). Intracellular Ca chelator BAPTA/AM and calmodulin antagonist W-7, but not Ca channel blocker nicardipine, significantly inhibited ET-3- or IRL 1620-induced cGMP generation. In cultured rat mesangial cells, ET-3 stimulated cGMP generation through NO in the presence of fetal calf serum, which was not inhibited by addition of BQ123-Na. In IMCD, ET-3 had no stimulative effect on cGMP generation. We conclude that ET-3 stimulates NO-induced cGMP generation through ETB receptor in glomerulus. This effect seems to be mediated through intracellular Ca/calmodulin, but not through Ca influx via L-type Ca channel. Mesangial cells can be a source of NO coupled to ETB receptor activation in glomerulus. From these results, mesangial ETB receptor may work to counteract the vasoconstrictive effect of endothelin caused via ETA receptor in glomerulus.     

The diagnostic value of the determination of cyclic 3',5'-adenosine monophosphate (cAMP) in urine]

Many hormones initiate their biologic actions by augmenting the intracellular concentrations of 3',5'-adenosine monophosphate (cyclic AMP). The nucleotide has been found in body fluids; its determination in plasma and urine can be performed by a rapid, simple and specific method: the cyclic AMP assay kit of the Radiochemical Centre (Amersham, England). The assay is based on the competition between unlabelled cAMP and a fixed quantity of the tritium labelled compound for binding to a bovine muscle protein which has a high specificity and affinity for cAMP. Different factors must be considered in evaluating the 24 h urinary content of the nucleotide: the renal or extrarenal origin of cAMP and the functional status of the kidneys. In basal conditions the urinary cAMP excretion is significantly correlated with creatinine excretion (n = 67; r = 0.47; p less than 0.001) thus confirming that the most part of cAMP excreted is derived from the plasma by glomerular filtration. Parathyroid hormone (PTH) stimulates adenylate cyclase predominantly in the renal cortex, whereas vasopressin (ADH) stimulated the enzyme in the medulla; thus PTH and ADH could increase the amount of cAMP in the urine from the renal source. In a case of diabetes insipidus and infusion of ADH caused a prompt rise in cAMP urinary excretion. In 5 normals an infusion of bovine synthetic parathyroid hormone caused an increased excretion of cAMP that preceded the phosphaturic response. An infusion of salmon synthetic calcitonin caused a rise in phosphate excretion and no increase in cAMP urinary content. As it concerns the two calciotopic hormones, PTH and CT, it is reasonable to assume that renal receptors are distinct. The 24 h urinary excretion of cAMP in 55 control subjects (3613 +/- 1460 D.S. n moles) was contrasted with the lower excretion in 25 elderly subjects (70-93 years: 1804 +/- 699 n moles), with the high cAMP excretion in a patient with hyperparathyroidism (that fell to normal values following removal of the parathyroid adenoma) and with the low cAMP excretion in patients with primary or surgical hypoparathyroidism. The mean 24 h cAMP excretion in patients with renal insufficiency was significantly decreased when compared to control subjects. These findings and recent reports confirm that the 24 h urinary output of cAMP may be considered an useful index of pharathyroid function in man.     

Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cyclic GMP and nitric oxide.

Voltage-gated Ca2+ channels contribute to the maintenance of contractile tone in vascular myocytes and are potential targets for vasodilating agents. There is no information available about their nature and regulation in human coronary arteries. We used the whole-cell voltage-clamp technique to characterize Ca2+-channel currents immediately after enzymatic dissociation and after primary culture of coronary myocytes taken from heart transplant patients. We recorded a dihydropyridine-sensitive L-type current in both freshly isolated and primary cultured cells. A T-type current was recorded only in culture. The L- (but not the T-) type current was inhibited by permeable analogues of cGMP in a dose-dependent manner. This effect was mimicked by the nitric oxide-generating agents S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine which increased intracellular cGMP. Methylene blue, known to inhibit guanylate cyclase, antagonized the effect of SNAP. Inhibitions by SNAP and cGMP were not additive and seemed to occur through a common pathway. We conclude that (a) L-type Ca2+ channels are the major pathway for voltage-gated Ca2+ entry in human coronary myocytes; (b) their inhibition by agents stimulating nitric oxide and/or intracellular cGMP production is expected to contribute to vasorelaxation and may be involved in the therapeutic effect of nitrovasodilators; and (c) the expression of T-type Ca2+ channels in culture may be triggered by cell proliferation.     

5'-Nor carbocyclic 5'-deoxy-5'-(isobutylthio)adenosine and a 2',3'-dideoxy-2',3'-didehydro derivative.

The inhibition of biochemical processes requiring S-adenosylmethionine as a co-factor have led to many nucleoside-based medicinal agents. Included in this group are 5'-deoxy-5'-(isobutylthio)adenosine (SIBA), a nucleoside with antiparasitic, antiviral and antiproliferative effects, and 5'-noraristeromycin, a carbocyclic-derived nucleoside with potent antiviral properties. This report brings together the structural components of these two compounds by describing both enantiomers of carbocyclic 5-nor SIBA (3 and 4). Owing to the recent interest in 2',3'-dideoxy-2',3'-didehydro nucleosides as antiviral agents, this derivative of 3 (5) is also described. All three compounds were screened against a variety of viruses and were found to be inactive at high concentrations or at limiting concentrations for the screening methods. The viruses subjected to 3-5 were herpes simplex virus types 1 and 2, human cytomegalovirus, vaccinia virus, vesicular stomatitis virus, respiratory syncytial virus, varicelIa zoster virus, coxsackie virus, parainfluenza-3 virus, sindbis virus, punta toro virus, reovirus-1, human immunodeficiency virus, influenza virus types A and B, adenovirus type 1 and measles virus. These results suggest that the C-5' methylene of the C-5' thio-based carbocyclic nucleosides is important for their antiviral properties.     

Change in brain guanosine 3',5'-monophosphate (cGMP) content by thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) causes a significant increase in rat cerebellar guanosine 3',5'-monophosphate (cGMP) content after parenteral administration. A smaller but still significant increase in cGMP was also observed in brain stem, whereas no significant changes were observed in cGMP in other gross brain regions or in adenosine 3',5'-monophosphate in any brain region. TRH also caused a similar increase in cerebellar cGMP content in hypophysectomized rats indicating that the increase is independent of an intact pituitary. The time course of cGMP elevation in the cerebellum after administration of 10 mg/kg of TRH i.v. showed a peak at 2.5 to 5.0 min followed by a less rapid decrease. The time course of TRH immunoreactivity in the same cerebellar homogenates roughly paralleled these changes. Only those TRH analogs which were previously shown to antagonize pentobarbital sleeping time in mice caused an elevation in cGMP content in the cerebellum. TRH also caused a significant increase in cerebellar cGMP in rats pretreated with phenobarbital and chlordiazepoxide. The TRH-induced increase in cerebellar cGMP was not affected by cerebellar climbing fiber lesions caused by 3-acetylpyridine nor blocked by haloperidol, suggesting that TRH acts by mechanisms different from harmaline or apomorphine in raising cerebellar cGMP.     

Circulating 3,3', 5'-triiodothyronine (reverse T3) in the human newborn.

Serum concentrations of 3,3',5'-triiodothyronine (reverse T3 rT3), 3,3',5-triiodothyronine (T), and thyroxine (T4) were measured in cord blood and invenous blood samples obtained between 2 h and 30 days of postnatal life from healthy full-term newborn infants. The mean serum rT3 concentration of (mean plus or minus SE) 151 plus or minus 12 ng per 100 ml in 18 cord blood samples was significantly higher than the level (41 plus or minus 2 ng per 100 ml) in 27 normal adult sera; the corresponding mean serum T4 of 12.7 plus or minus 0.8 mug per 100 ml in cord blood also was significantly higher than that (8.6 plus or minus 1.9 mug per 100 ml) in 108 normal adults. By contrast, the mean serum T3 concentration in 15 cord blood samples, 24 plus or minus 3 mg per 100 ml, was significantly lower than the value of 126 plus or minus 3.2 ng per 100 ml measured in 108 normal adults. At 4 h of age the mean serum rT3 concentration (165 plus or minus 13 ng per 100 ml) in six newborns was 4ot significantly different from that in paired cord blood samples (194 plus or minus 25 ng per 100 ml); on the other hand, whenever, studied, the mean serum T3 and T4 levels were significantly higher at 4 h than at birth. The failure of serum rT3 concentrations to rise after delivery in response to the early neonatal thyrotropin (TSH) surge and at a time when serum T3 and T4 levels increase significantly prompted a study of the rT3 response to 10 IU of intramuscular TSH in three healthy adult subjects. Just as in the newborns, serum rT3 failed to rise appreciably in these subjects, even though serum T3 and T4 showed the expected increments. Serum rT3 concentrations in 1-4 day-old newborn infants did not differ significantly from values in the cord blood but were significantly lower in older neonates. The mean serum rT3 level in 5-7-day-old infants was higher than that in normal adults, but in 9-11 day and 20-30-day-old infants, mean rT3 values were statistically similar to the adult value. The mean serum T3 concentrations in neonates between 1-30 days old were either higher than or comparable to the values of normal adults. The mean serum T4 concentrations in neonates between birth and 30 days of age were significantly higher than the mean adult level. The mean serum rT3 to T4 ratios (rT3/T4) were elevated in 1-4-day-old neonates; the values in older neonates were similar to those in adults. These results suggest that (a) factors other than TSH are important modulators of serum rT3 in man; (b) high serum rT3 concentration in the newborn becomes comparable to that in the normal adult by 9-11 days of neonatal life.