Selective inhibition of rat and human cardiac guanylate cyclase in vitro by doxorubicin (adriamycin): possible link to anthracycline cardiotoxicity.

The cardiotoxicity of anthracycline antibiotic anti-tumor agents is well-described but the molecular basis of the cardiotoxicity is not understood. We examined the effect of doxorubicin (Adriamycin) on the activity of guanylate cyclase (E.C. 4.6.1.2), the enzyme catalyzing the production of guanosine 3′,5′-monophosphate, from rat heart, liver, lung, kidney, and spleen. Doxorubicin produced a decrease in cardiac guanylate cyclase activity over the concentration range 0.4 to 2 mm but was without effect, or slightly stimulated, guanylate cyclase from the other tissues. Daunorubicin (Daunomycin), a related, cardiotoxic anthracycline antibiotic also decreased cardiac guanylate cyclase activity over the concentration range 0.8 to 4 mm. Other antibiotic anti-tumor agents which are not cardiotoxic, including streptonigrin, porfiromycin, and mitomycin C did not decrease cardiac guanylate cyclase activity. Doxorubicin, 1 mm and 2 mm, and daunorubicin, 4 mm decreased cardiac guanylate cyclase approximately 50% in experiments utilizing guanylate cyclase prepared from human heart. The data suggests that some aspects of anthracycline cardiotoxicity may be related to altered cardiac guanylate cyclase activity.     

Bromocriptine enhances guanylate cyclase activity

Bromocriptine and its parent compound alpha-ergocryptine were investigated with respect to their ability to interact with the guanylate cyclase (E.C.4.6.1.2)-cyclic GMP system in vitro in the rat pituitary and ovary. Both bromocriptine and alpha-ergocryptine enhanced guanylate cyclase two- to threefold in both of these tissues over a concentration range of 1 nM to 1 microM. Since bromocriptine is thought to be a dopamine agonist in the pituitary, dopamine's effects on guanylate cyclase were also tested. Dopamine caused a twofold enhancement of guanylate cyclase activity in the pituitary and ovary. When bromocriptine and dopamine were used in combination, bromocriptine had to be in equal or a greater concentration with respect to dopamine in vitro to enhance guanylate cyclase activity. These findings suggest that bromocriptine's effect at the level of the pituitary and ovary may be mediated through enhancement of guanylate cyclase activity.     

Modulation of guanylate cyclase by lipoxygenase inhibitors

Drugs that inhibit endothelium-dependent relaxation were tested to determine their effect on soluble guanylate cyclase purified from dog aorta. Basal, arachidonic acid (10(-5) M)-stimulated, and nitroprusside (5 X 10(-5) M)-stimulated guanylate cyclase activities were inhibited by methylene blue and the lipoxygenase inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid. The effective inhibitory doses were in the range of those that have been reported to inhibit endothelium-dependent relaxation. Other compounds known to inhibit endothelium-dependent relaxation had little or no effect on guanylate cyclase activity. Basal guanylate cyclase activity was more resistant to inhibition than were activated states of the enzyme. The data suggest that reported inhibition of endothelium-dependent relaxation by some lipoxygenase inhibitors may be the result, at least in part, of their direct effect on guanylate cyclase activity.     

Long-term and short-term models for studying anthracycline cardiotoxicity and protectors

The clinical importance of the cardiotoxicity of anthracyclines requires the availability of preclinical models able to predict the cardiotoxicity of novel anthracycline analogs in reference to doxorubicin or of cardioprotectors aimed at circumventing the deleterious effects of these drugs. The reference model has been defined long ago and has proven its validity. Weanling rabbits given weekly injections of doxorubicin for 4 months developed a cardiomyopathy, which can be assessed from a clinical and pathological point of view. Models in other animals such as rats or mice were similarly implemented, also with long-term exposures to the drug, resulting in cardiac failure and severe pathological alterations, which could be graded for comparison. Starting from the evidence that the damage caused by anthracyclines on cardiomyocytes was immediate after each injection and that the functional efficiency of the myocardium should be affected long before the morphological alterations become detectable, we developed a short-term model studying the cardiac performances of isolated perfused hearts of rats that had been treated within 12 days by repetitive administrations of the molecule(s) to be tested. This model provided the data expected from clinical experience: epirubicin appeared less cardiotoxic than doxorubicin; liposomal formulations appeared less cardiotoxic than free drug formulations; dexrazoxane strongly protected against doxorubicin cardiotoxicity. We were then able to show that paclitaxel could potentialize doxorubicin cardiotoxicity, but that docetaxel did not so; or that a high dose of dexrazoxane brought significantly higher protection than a conventional dose. Based upon these contributions, we can encourage the use of the short-term model of isolated perfused rat heart to screen the preclinical cardiotoxicity of anthracycline molecules, formulations and combinations.     

The interval-force relationship: a technique for evaluating the cardiac toxicity of anthracycline analogs.

The most serious side effect of the anthracycline derivatives is dose-related cardiac toxicity induced during repeated administration. An in vitro method is described which assesses the interval-force relationship in evaluating the contractility of the rat and rabbit heart. Repeated administration of Adriamycin resulted in a progressive decrease in contractility which correlated closely with the cumulative dose administered. This model offers a reliable method to evaluate the effect of new anthracycline analogs on the heart and to study the potential of other drugs or agents to protect against cardiac toxicity.     

Effect of in vitro organic nitrate tolerance on relaxation, cyclic GMP accumulation, and guanylate cyclase activation by glyceryl trinitrate and the enantiomers of isoidide dinitrate

Previously, it was shown that the D enantiomer of isoidide dinitrate was 10-fold more potent than the L enantiomer and 10-fold less potent than glyceryl trinitrate for stimulating cyclic GMP accumulation and relaxation of isolated rat aorta. In the present study, these organic nitrates were tested for their ability to induce tolerance to organic nitrate-induced relaxation, cyclic GMP accumulation, and guanylate cyclase activation in rat aorta in vitro. To compensate for the differences in vasodilator potency, tolerance was induced by incubating isolated rat aorta with concentrations of organic nitrates 1,000-fold greater than the EC50 for relaxation. Under these conditions, the EC50 for relaxation was increased significantly for each organic nitrate and to a similar degree on subsequent reexposure. These data suggest that the potential for inducing in vitro tolerance to relaxation was the same for the three organic nitrates tested. When activation of soluble guanylate cyclase by these compounds was assessed, the enantiomers of isoidide dinitrate were equipotent, but less potent than glyceryl trinitrate, suggesting that the site of enantioselectivity is not guanylate cyclase itself. In blood vessels made tolerant to organic nitrates by pretreatment with glyceryl trinitrate, vasodilator activity, cyclic GMP accumulation, and guanylate cyclase activation were attenuated on reexposure to each organic nitrate. In addition, differences in the potency of the three organic nitrates and the enantioselectivity of isoidide dinitrate for relaxation were abolished in tolerant tissue, whereas the potency difference between glyceryl trinitrate and isoidide dinitrate for activation of guanylate cyclase was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogens and progesterone increase fetal and maternal guanylate cyclase activity

Since both estrogens and cyclic guanosine 3',5'-monophosphate stimulate protein synthesis, the objective of the present investigation was to determine if estrogens and their precursors might have part of their mechanism of action through stimulation of guanylate cyclase (E.C.4.6.1.2), the enzyme that catalyzes the conversion of guanosine triphosphate to cyclic guanosine 3',5'-monophosphate. The precursors of estrogen synthesis originate from cholesterol. Cholesterol itself had no effect on guanylate cyclase activity. The precursors of estrogen synthesis generated from cholesterol, namely, progesterone, 17 alpha-OH-progesterone, androstenedione, pregnenolone, 17 alpha-OH-pregnenolone, and dehydroepinandrosterone, however, caused a 2- to 3-fold enhancement of fetal and maternal guinea pig hepatic and uterine guaynlate cyclase activity at a concentration of 1 microM. In comparative studies, similar effects were seen on immature female Sprague-Dawley rat hepatic and uterine guanylate cyclase activity. Estrone, estradiol-17 beta, estriol, and the synthetic estrogen, diethylstilbestrol, enhanced guanylate cyclase activity in the same tissues 2- to 3- fold at the 1 microM concentration. Dose-response relationships revealed that these estrogens and their precursors had their maximal effect at 0.001 microM. Estradiol-17 alpha also enhanced uterine guanylate cyclase activity, but a 1000-fold greater concentration compared to the other estrogens was necessary to show any significant effect. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of estrogens and their precursors.     

Guanylate cyclase activity in fetal, neonatal and adult rat heart.

Guanosine 3′, 5′-monophosphate is thought to be an important factor in promoting cell growth. We, therefore, measured the activity of guanylate cyclase, the enzyme which in the developing rat heart catalyzes the production of guanosine 3′, 5′-monophosphate. Cardiac guanylate cyclase activity was found to be increased to 405 ± 44 pmol accumulated/10/min/mg protein in utero and maintained this level of activity for the first 3 weeks after birth. By 28 days, cardiac guanylate cyclase activity had decreased to 165 ± 7 pmol accumulated/10 min/mg protein which paralleled the adult mother cardiac guanylate cyclase activity of 173 ± 9 pmol accumulated/10 min/mg protein. This period of maximal activity of cardiac guanylate cyclase corresponds to the period of rapid growth of the heart and the decreased activity approximates the period of time when cardiac DNA synthesis declines.     

Production and characterization of monoclonal antibodies to soluble rat lung guanylate cyclase.

Four monoclonal antibodies to rat lung soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2] have been produced by fusing spleen cells from immunized BALB/c mice with SP-2/0 myeloma cells. The antibodies were detected by their ability to bind immobilized guanylate cyclase and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibody. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid containing 2-5 mg of antibody per ml. The four antibodies obtained had titers of between 1:1580 and 1:3160 but were detectable at dilutions greater than 1:20,000. Soluble guanylate cyclase from several rat tissues were crossreactive with the four monoclonal antibodies, suggesting that the soluble enzyme from different rat tissues is antigenically similar. The antibodies also recognized soluble lung enzyme from rat, beef, and pig, while enzyme from rabbit was not crossreactive and mouse enzyme was recognized by only one of the antibodies. Particulate guanylate cyclase from a number of tissues had only minimal crossreactivity with the antibodies. Immunoprecipitated guanylate cyclase retained catalytic activity, could be activated with sodium nitroprusside, and was inhibited by cystamine. None of the antibodies were inhibitory under the conditions examined. These antibodies will be useful probes for the study of guanylate cyclase regulation and function under a variety of physiological conditions.     

Direct effect of glibenclamide on guanylate cyclase activity in the rat in vitro

Summary Glibenclamide enhanced the activity in the rat of guanylate cyclase in a number of extra-pancreatic tissues. Thus, glibenclamide enhanced guanylate cyclase activity in vitro two- to threefold in liver, kidney, heart, spleen and colon at a concentration of 1 mol/l. Dose-response curves of glibenclamide on hepatic guanylate cyclase revealed that more than half-maximal stimulation was observed at a concentration as low as 10 nmol/l (p< 0.001) and no stimulation of guanylate cyclase was seen when the concentration was decreased to 1 nmol/l. Maximal enhancement was seen at 100 nmol/l of glibenclamide. Varying the concentration of the guanylate cyclase co-factor manganese had no effect on the glibenclamide enhancement of guanylate cyclase. In addition to the increased insulin receptors found recently in monocytes and fibroblasts, the present findings may help explain the extra-pancreatic effects of glibenclamide and possibly of other sulphonylurea drugs.     

Testosterone and its precursors and metabolites enhance guanylate cyclase activity.

Both testosterone and cyclic GMP stimulate DNA synthesis. Because cyclic GMP and testosterone seem to have similar actions, the objective of this investigation was to determine if testosterone and its precursors might have part of their mechanism of action through stimulation of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2], the enzyme that catalyzes the formation of cyclic GMP from GTP. The precursors--namely, progesterone, pregnenolone, 17 alpha-progesterone, 17 alpha-hydroxypregnenolone, androstenedione, and dehydroepiandrosterone--caused a 2- to 3 1/2-fold enhancement of guanylate cyclase activity in rat liver, kidney, skeletal muscle, and ventral prostate at a concentration of 1 microM. These precursors are generated from cholesterol, which had no effect itself on guanylate cyclase activity. Testosterone, 19-nortestosterone, 17-methyltestosterone, and 5 alpha-dihydrotestosterone enhanced guanylate cyclase activity 2- to 5-fold in the same tissues at 1 microM. Etiocholanolone, androsterone, and epiandrosterone, metabolites of testosterone metabolism, enhanced guanylate cyclase activity 1 1/2- to 2-fold at this same concentration. Dose-response relationships revealed that testosterone and its precursors and metabolites had their maximal effect at 1 microM but still had some effect at 0.001 microM. The data in this investigation suggest that the guanylate cyclase-cyclic GMP system plays a role in the mechanism of action of testosterone and its precursors.     

Doxorubicin-induced ventricular arrhythmia treated by implantation of an automatic cardioverter-defibrillator.

Anthracyclines are a group of potent antitumour agents and cardiotoxicity is an important factor limiting their therapeutic effectiveness. Although cardiomyopathy is the most widely recognized type of cardiotoxic reaction, early arrhythmia following anthracycline administration may be of clinical significance as well. We report a case of ventricular tachycardia causing cardiac arrest in a female treated with doxorubicin as adjuvant therapy of breast cancer. Due to recurrence of the arrhythmia and a desire to continue chemotherapy, an automatic cardioverter-defibrillator was implanted with excellent effect.     

Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations

Nitric oxide gas (NO) increased guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity in soluble and particulate preparations from various tissues. The effect was dose-dependent and was observed with all tissue preparations examined. The extent of activation was variable among different tissue preparations and was greatest (19- to 33-fold) with supernatant fractions of homogenates from liver, lung, tracheal smooth muscle, heart, kidney, cerebral cortex, and cerebellum. Smaller effects (5- to 14-fold) were observed with supernatant fractions from skeletal muscle, spleen, intestinal muscle, adrenal, and epididymal fat. Activation was also observed with partially purified preparations of guanylate cyclase. Activation of rat liver supernatant preparations was augmented slightly with reducing agents, decreased with some oxidizing agents, and greater in a nitrogen than in an oxygen atmosphere. After activation with NO, guanylate cyclase activity decreased with a half-life of 3-4 at 4 degrees but re-exposure to NO resulted in reactivation of preparations. Sodium azide, sodium nitrite, hydroxylamine, and sodium nitroprusside also increased guanylate cyclase activity as reported previously. NO alone and in combination with these agents produced approximately the same degree of maximal activation, suggesting that all of these agents act through a similar mechanism. NO also increased the accumulation of cyclic GMP but not cyclic AMP in incubations of minces from various rat tissues. We propose that various nitro compounds and those capable of forming NO in incubations activate guanylate cyclase through a similar but undefined mechanism. These effects may explain the high activities of guanylate cyclase in certain tissues (e.g., lung and intestinal mucosa) that are exposed to environmental nitro compounds.     

Nitroimidazole derivatives inhibit anterior pituitary cell function apparently by a direct effect on the catalytic subunit of the adenylate cyclase holoenzyme

Nitroimidazole derivatives dose-dependently decreased basal and CRF-stimulated ACTH release, basal and GRF-stimulated rat GH release, and basal rat PRL release in primary cultures of rat anterior pituitary cells. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with the nitroimidazole derivatives. Miconazole, econazole, isoconazole, clotrimazole, and bifonazole had similar or more pronounced effects on anterior pituitary function compared to ketoconazole, whereas metronidazole and etomidate were less effective. The positive correlation between the number of phenylated side-chains or phenolic rings of the imidazole molecule and the efficacy to inhibit activity on pituitary hormone secretion suggests a structure-activity relationship of these compounds. The effects of the nitroimidazole derivatives on anterior pituitary hormone release and biosynthesis were mediated by cAMP. Thus, basal and CRF-, cholera toxin-, and forskolin-stimulated adenylate cyclase activities in rat anterior pituitary cell membranes determined by cAMP formation were suppressed by the nitroimidazole derivatives. Pertussis toxin did not diminish the nitroimidazole derivative effect on cAMP formation. The adenylate cyclase inhibitory effect of these substances was independent of the presence of GTP in the assay system, underlining a direct effect on the catalytic subunit. In addition, basal and forskolin-stimulated cAMP generation in membranes of S49 lymphoma cyc-variants, which lack a functional Gs protein, was efficiently suppressed (by up to 90%) by the nitroimidazole derivatives. In conclusion, ketoconazole and other nitroimidazole derivatives inhibit anterior pituitary hormone synthesis and secretion apparently by a direct effect on the catalytic subunit of the adenylate cyclase system.     

Activation of intestinal guanylate cyclase by heat-stable enterotoxin of Escherichia coli: studies of tissue specificity, potential receptors, and intermediates

Heat-stable enterotoxin (ST) of Escherichia coli increased guanylate cyclase activity in homogenates of rat and rabbit intestinal mucosa and stimulated intestinal fluid secretion in suckling mice. The ST effect on guanylate cyclase was dose-dependent, occurred without a time lag, and was confined to the particulate fraction. ST activation of guanylate cyclase was tissue-specific; ST did not alter activity of soluble or particulate rat liver, lung, heart, kidney, or cerebral cortex enzyme. The ST activity on guanylate cyclase and secretion was methanol-soluble and alkali-labile, and its effects were not altered by phentolamine, propranolol, or atropine. Monosialoganglioside did not reduce ST-induced secretion. However, indomethacin and butylated hydroxyanisole decreased the ST effect on both guanylate cyclase and secretion. Fluid secretion with ST sppears to result from specific activation of particulate intestinal guanylate cyclase. While adrenergic and cholinergic events are probably not involved in this process, the effects of ST may be mediated through prostaglandin synthesis or oxidative mechnanisms.     

Trichostatin A accentuates doxorubicin-induced hypertrophy in cardiac myocytes

Histone deacetylase inhibitors represent a new class of anticancer therapeutics and the expectation is that they will be most effective when used in combination with conventional cancer therapies, such as the anthracycline, doxorubicin. The dose-limiting side effect of doxorubicin is severe cardiotoxicity and evaluation of the effects of combinations of the anthracycline with histone deacetylase inhibitors in relevant models is important. We used a well-established in vitro model of doxorubicin-induced hypertrophy to examine the effects of the prototypical histone deacetylase inhibitor, Trichostatin A. Our findings indicate that doxorubicin modulates the expression of the hypertrophy-associated genes, ventricular myosin light chain-2, the alpha isoform of myosin heavy chain and atrial natriuretic peptide, an effect which is augmented by Trichostatin A. Furthermore, we show that Trichostatin A amplifies doxorubicin-induced DNA double strand breaks, as assessed by γH2AX formation. More generally, our findings highlight the importance of investigating potential side effects that may be associated with emerging combination therapies for cancer.     

Receptor regulation of atrial natriuretic factor

Two atrial natriuretic factor (ANF) receptor subtypes are present in vascular smooth muscle cells: the B receptors (or biologically active) coupled to a guanylate cyclase and the C receptors (clearance) representing 95% of the total number of ANF binding sites but noncoupled to a guanylate cyclase. Using binding experiments with 125I-ANF and measurement of cGMP production stimulated by ANF, we were able to demonstrate that ANF receptors are sensitive to homologous (induced by ANF) and heterologous regulation (induced by angiotensin II, AII) in rat cultured vascular smooth muscle cells. The effect of the two hormones showed marked differences, in their time course, their reversibility and their consequence on guanylate cyclase activity. Although both ANF and AII reduced the total number of ANF binding sites after 18 h, ANF induced a desensitization of the guanylate cyclase whereas AII elicited a potentialization of this system. From these results, we have concluded that in vascular cells B receptors are sensitive to homologous regulation and C receptors are sensitive to heterologous regulation by AII. This also highlights a specific interaction between ANF and AII at the receptor level.     

The role of Ca2+ and calmodulin in the regulation of atrial natriuretic peptide-stimulated guanosine 3',5'-cyclic monophosphate accumulation by isolated mouse Leydig cells

We have investigated the role of Ca2+ and calmodulin in the stimulation of cGMP formation by mouse Leydig cells in response to rat atriopeptin-II (rAP-II). Removal of extracellular Ca2+ had no influence on the levels of cGMP accumulated by the cells stimulated with rAP-II. The amounts of testosterone produced by unstimulated and rAP-II-stimulated cells were, however, reduced by 50% in the absence of Ca2+ from the incubation medium. Addition of ionomycin to the Leydig cells led to a dose-related inhibition of rAP-II-stimulated cGMP formation, but the basal cGMP level was not affected. These experiments were carried out in the presence of a phosphodiesterase inhibitor. The inhibitory effect of ionomycin was absolutely dependent upon the presence of Ca2+ in the medium. The guanylate cyclase activity required the presence of a cation, and Mn2+, Mg2+, or Ca2+ could function as the required cation. There was no direct inhibition of the cyclase activity by Ca2+ up to as high a concentration as 8 mM. Furthermore, three structurally unrelated calmodulin antagonists, W7, trifluoperazine, and calmidazolium, but not W5, caused a dose-related inhibition of rAP-II-stimulated cGMP accumulation by the cells. The inhibitory effect of calmodulin antagonists was not exerted directly at the level of guanylate cyclase activity, since the particulate enzyme was not inhibited by any of these drugs. We conclude, therefore, that extracellular Ca2+ is not essential for rAP-II-mediated stimulation of cGMP formation by mouse Leydig cells, at least under the short term incubation conditions used. An excessive ionophoretic influx of Ca2+ into the cells impairs the ability of rAP-II to stimulate cGMP formation. Therefore, it appears that a finely regulated level of intracellular Ca2+ is required for optimal activation of atrial natriuretic peptide-responsive guanylate cyclase in mouse Leydig cells, and calmodulin plays an important role in this process.     

Evidence for a correlation between nitric oxide formation by cleavage of organic nitrates and activation of guanylate cyclase

According to our present understanding organic nitrates like glycerine trinitrate mediate their pharmacological effect by an intracellular stimulation of the enzyme guanylate cyclase (E.C. 4.6.1.2.) [1, 10]. The exact molecular mechanism underlying the process of enzyme activation is still a matter of controversial discussion. But there is general agreement in literature about the fact that organic nitrate compounds are able to activate the enzyme guanylate cyclase only in the presence or by the interaction of the amino acid cysteine [3, 5]. The stimulatory activity of nitric oxide-containing compounds may be due, at least in part, to the formation of active, unstable intermediate S-nitrosothiols, i.e. S-nitrosocysteine in case of the organic nitrates [7]. According to Craven and DeRubertis [2], the active intermediates of guanylate cyclase stimulation are represented by nitric oxide-heme complexes. There is, however, substantial evidence that the organic nitrates have to be cleaved before they become biologically active. During the transformation which takes place in the presence of cysteine or by means of enzymatic catalysis, nitric oxide radicals are reductively split off the molecule from which (via the intermediate formation of salpetric acid) the nitric oxide is liberated as the essential stimulatory agent. In this study we examined the transformation of glycerine trinitrate and other organic nitrates under the influence of different thiols and a purified soluble rat liver guanylate cyclase preparation. At the same time the stimulation of guanylate cyclase in the presence of the thiols mentioned was quantitatively estimated. Only in case of cysteine did we find a strict correlation between the liberation of nitric oxide from different organic nitrates and the degree of enzyme activation. Several other thiols were also able to liberate nitric oxide, but surprisingly enough, there was no equivalent stimulation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)