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PubMed1983

Ligand exchange reactions of diol dehydrase-bound cobalamins and the effect of the nucleoside binding.

The inactive complex of diol dehydrase with hydroxocobalamin was resolved by treatment with SO2-3, followed by dialysis to remove SO2-3, giving the apoenzyme which was reconstitutable into catalytically active holoenzyme upon addition of adenosylcobalamin ("re-activation"). Spectral evidence showed that the enzyme-bound hydroxocobalamin undergoes a Co beta-ligand exchange reaction forming sulfitocobalamin. Sulfitocobalamin was bound to diol dehydrase only loosely, and therefore dissociated from the enzyme. In contrast, neither the enzyme-hydroxocobalamin-5'-deoxyadenosine nor the enzyme-hydroxocobalamin-adenosine complex was resolved and thus re-activated by this procedure. It was shown spectroscopically that the hydroxocobalamin in these complexes does not react with SO2-3, or even with CN-, indicating that the OH group in the Co beta-position was blocked spatially by these enzyme-bound nucleosides. Neither O2-inactivated holoenzyme nor the holoenzyme inactivated suicidally by glycerol or 1,2-ethanediol during dehydration reaction was also re-activated by the same procedure. The complex of the enzyme with cyanocobalamin or methylcobalamin was not resolvable by the SO2-3 treatment. This was because these cobalamins bound to the enzyme were not subject to a ligand exchange reaction with SO2-3.

Cobamides↗

PubMed1995

Effects of alternating current iontophoresis on drug delivery.

OBJECTIVE: The duration of direct current (DC) iontophoresis is limited to 10- to 15-minute periods because of electrochemical burns from hydrogen and hydroxide ions generated by the DC current. A new iontophoretic device, the Lectro Patch, uses a low-frequency alternating current (AC). AC current is theorized to generate H+ ions during one phase and OH- when the current reverses polarity, thus possibly neutralizing pH changes and avoiding burns. This study examined this possibility and evaluated drug delivery with AC iontophoresis, using hydroxocobalamin. DESIGN: A known amount of hydroxocobalamin dissolved in 6mL of water was loaded in Lectro Patches, two of which were then taped on the forearms of 10 patient volunteers. One patch was activated to deliver drug by AC iontophoresis. The second patch was not activated and served as a control for delivery by diffusion. Trials were run for 2 and 4 hours, with both 1,000 micrograms/mL and 2,000 micrograms/mL concentrations. SETTING: Study was conducted with inpatients in an extended care setting using volunteers. MAIN OUTCOME MEASURES: Amounts of hydroxocobalamin remaining in the Lectro Patches after iontophoresis were assayed by spectrophotometry. Data were analyzed by ANOVA. RESULTS: No burns occurred. Significantly greater losses occurred with 4 hours of iontophoresis than with 2 hours (p < 0.05). There was no significant effect of changing the concentration of hydroxocobalamin. CONCLUSIONS: AC iontophoresis avoids electrochemical burns; charged drugs can be delivered by AC iontophoresis; and delivery of drug increases with duration of application.

Drug Delivery Systems↗

PubMed1977

Conversion of hydroxo(aquo) cobalamin to sulfitocobalamin in the absence of light: a reaction of importance in the identification of the forms of vitamin B12, with possible clinical significance.

During determinations of the forms of vitamin B12 in foods and human tissues several samples yielded a growth zone on bioautography which was distinct from those due to methylcobalamin, adenosylcobalamin, hydroxocobalamin, or cyanocobalamin. The material responsible for this growth zone was identified as sulfitocobalamin and the mechanisms in its formation from hydroxocobalamin involve absence of light and the presence of bisulphite ions derived from atmospheric sulfur dioxide or indigenous sulfite ions. The formation of sulfitocobalamin during the extraction of cobalamins from foods and tissues can be prevented by reception and homogenization of the material in an ammonia buffer instead of water; the hydroxocobalamin is then converted to ammonia cobalamin, which is more resistant to attack by (bi)sulfite ions. The conversion of hydroxo(aquo)cobalamin to sulfitocobalamin in the dark can be rapid, and materials for analysis should be placed in the ammonia buffer before darkroom work is begun. The rapid conversion of hydroxo(aquo)cobalamin to sulfitocobalamin in the dark raises the possibility that hydroxocobalamin in foods may be converted to the less well absorbed sulfitocobalamin in the upper gastrointestinal tract.

Animals↗

PubMed2002

Relaxation to bradykinin in bovine pulmonary supernumerary arteries can be mediated by both a nitric oxide-dependent and -independent mechanism.

1. The aim of the present study was to determine the relative contribution of prostanoids, nitric oxide and K(+) channels in the bradykinin-induced relaxation of bovine pulmonary supernumerary arteries. 2. In endothelium-intact, but not denuded rings, bradykinin produced a concentration-dependent relaxation (pEC(50), 9.6+/-0.1), which was unaffected by the cyclo-oxygenase inhibitor indomethacin. The nitric oxide scavenger hydroxocobalamin (200 micro M, pEC(50), 8.5+/-0.2) and the nitric oxide synthase inhibitor L-NAME (100 micro M, pEC(50), 8.9+/-0.1) and the combination of L-NAME and hydroxocobalamin (pEC(50), 8.1+/-0.2) produced rightward shifts in the bradykinin concentration response curve. 3. The guanylyl cyclase inhibitor ODQ (10 micro M, pEC(50), 9.6+/-0.4) did not affect the response to bradykinin. 4. Elevating the extracellular [K(+)] to 30 mM did not affect the response to bradykinin but abolished the response when ODQ or L-NAME was present. 5. The K(+) channel blocker apamin (100 nM), combined with charybdotoxin (100 nM), produced a small reduction in the maximum response to bradykinin but they abolished the response to bradykinin when ODQ, L-NAME or hydroxocobalamin were present. Apamin (100 nM) combined with iberiotoxin (100 nM) also reduced the response to bradykinin in the presence of hydroxocobalamin or L-NAME. 6. The concentration response curve for sodium nitroprusside-induced relaxation was abolished by ODQ (10 micro M) and shifted to the right by apamin and charybdotoxin. 7. These studies suggest that in bovine pulmonary supernumerary arteries bradykinin can stimulate the formation of nitric oxide and activate an EDHF-like mechanism and that either of these pathways alone can mediate the bradykinin-induced relaxation. In addition nitric oxide, acting through guanylyl cyclase, can activate an apamin/charbydotoxin-sensitive K(+) channel in this tissue.

Animals↗

PubMed2003

Mediators of bradykinin-induced vasorelaxation in human coronary microarteries.

To investigate the mediators of bradykinin-induced vasorelaxation in human coronary microarteries (HCMAs), HCMAs (diameter approximately 300 microm) obtained from 42 heart valve donors (20 men and 22 women; age range, 3 to 65 years; mean age, 46 years) were mounted in Mulvany myographs. In the presence of the cyclooxygenase inhibitor indomethacin, bradykinin relaxed preconstricted HCMAs in a concentration-dependent manner. N(G)-nitro-L-arginine methyl ester and ODQ (inhibitors of nitric oxide [NO] synthase and guanylyl cyclase, respectively) and the NO scavenger hydroxocobalamin, either alone or in combination, shifted the bradykinin concentration-response curve to the right. Removal of H2O2 (with catalase), inhibition of cytochrome P450 epoxygenase (with sulfaphenazole or clotrimazole) or gap junctions (with 18alpha-glycyrrhetinic acid or carbenoxolone), and blockade of large- (BK(Ca)) and small- (SK(Ca)) conductance Ca2+-dependent K+ channels (with iberiotoxin and apamin), either alone or in addition to hydroxocobalamin, did not affect bradykinin. In contrast, complete blockade of bradykinin-induced relaxation was obtained when we combined the nonselective BK(Ca) and intermediate-conductance (IK(Ca)) Ca2+-dependent K+ channel blocker charybdotoxin and apamin with hydroxocobalamin. Charybdotoxin plus apamin alone were without effect. Inhibition of inwardly rectifying K+ channels (K(IR)) and Na+/K+-ATPase (with BaCl2 and ouabain, respectively) shifted the bradykinin concentration-response curve 10-fold to the right but did not exert an additional effect in the presence of hydroxocobalamin. In conclusion, bradykinin-induced relaxation in HCMAs depends on (1) the activation of guanylyl cyclase, K(IR), and Na(+)/K(+)-ATPase by NO and (2) IK(Ca) and SK(Ca) channels. The latter are activated by a factor other than NO. This factor is not a cytochrome P450 epoxygenase product or H2O2, nor does it depend on gap junctions or BK(Ca) channels.

8,11,14-Eicosatrienoic Acid↗

PubMed1980

Inherited methylmalonyl CoA mutase apoenzyme deficiency in human fibroblasts: evidence for allelic heterogeneity, genetic compounds, and codominant expression.

We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut degrees mutants. Those with detectable residual activity in cell extracts ( approximately 0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut(-) mutants. The mutant enzyme in the mut(-) mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (K(m)) for adenosylcobalamin in vitro, a normal K(m) for methylmalonyl CoA, and a strikingly reduced thermal stability at 45 degrees C relative to control. Mutase from one mut(-) mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.To detect heterozygous carriers of mutant mut alleles, we compared mutase activity in fibroblast extracts from four controls with that from eight parents of either mut degrees or mut(-) mutants. After cell growth in either unsupplemented or hydroxocobalamin-supplemented medium, activity in cell lines from heterozygotes was reduced to 47 or 37% of the mean control activities, respectively. We also examined the effect of adenosylcobalamin concentration on reaction kinetics of mutase from heterozygote cell lines. All four cell lines from parents of mut(-) mutants exhibited complex enzyme kinetics; approximately 80% of mutase activity demonstrated a K(m) indistinguishable from control, whereas a smaller component of activity exhibited a K(m) similar to the abnormal K(m) expressed by the mut(-) propositus in each family. In two families with a mut degrees propositus, mutase from three of the four parents exhibited only the normal K(m) for adenosylcobalamin, whereas mutase from one parent displayed complex kinetics, indicating expression of both a normal allele (mut(+)) and a mutant allele with an abnormal K(m). From these studies, we conclude that mut mutants reflect mutations at the autosomal gene locus for the methylmalonyl CoA mutase apoenzyme; that mut degrees , mut(-), and mut(+) alleles at this locus are codominantly expressed; and that some mut mutants may be genetic compounds, inheriting two different mut degrees or mut(-) alleles from their parents.

Alleles↗

PubMed2002

Involvement of gap junctions in bradykinin-induced relaxation of bovine pulmonary supernumerary arteries before and after inhibition of nitric oxide/guanylate cyclase.

This study evaluated the possible contribution of gap junctions to the nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated responses elicited by bradykinin in bovine pulmonary supernumerary arteries. In artery rings with an intact endothelium and treated with the cyclo-oxygenase inhibitor indomethacin (10 microM), bradykinin (100 pM-1 microM) produced a concentration-dependent relaxation [-logEC(50) (pEC(50)), 9.6+/-0.2; maximum relaxation ( R (max)), 89.7+/-14.8%; n =6]. The NO synthase inhibitor N (G)-nitro-L-arginine methyl ester (L-NAME; 100 microM) and the NO scavenger hydroxocobalamin (200 microM) each produced a rightward shift in the bradykinin concentration-response curve [pEC(50): L-NAME, 8.9+/-0.1 ( n =6; P <0.01); hydroxocobalamin, 8.3+/-0.2, ( n =6; P <0.001)]. However, the soluble guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10 microM) did not significantly alter the response to bradykinin (pEC(50) 9.4+/-0.2; n =9). The gap junction inhibitor carbenoxolone (100 microM) did not affect the relaxation produced by bradykinin (pEC(50), 9.7+/-0.1; R (max), 100+/-3.2%; n =6), but it significantly depressed R (max) when L-NAME, hydroxocobalamin or ODQ was present. Further, carbenoxolone produced a rightward shift in the bradykinin concentration-response curve in the presence of ODQ (8.4+/-0.1; n =6, P <0.01). The data suggest that, in bovine pulmonary supernumerary arteries, gap junctions may, in part, facilitate the EDHF-mediated response, but not the NO-mediated response, to bradykinin. However, the additional involvement of an unidentified endothelial relaxing factor cannot be excluded.

Animals↗

PubMed1993

Use of vitamin B12 in the treatment and prevention of nitroprusside-induced cyanide toxicity.

OBJECTIVE: To review the mechanism of action, safety, and efficacy of hydroxocobalamin in the treatment and prevention of nitroprusside-induced cyanide toxicity. DATA SOURCES: English and foreign-language journal articles and reference texts identified from Index Medicus. Both animal and human studies were included. DATA SYNTHESIS: High-dose or prolonged therapy with nitroprusside in patients with hepatic or renal dysfunction increases the risk for nitroprusside-induced cyanide or thiocyanate toxicity, respectively. Hydroxocobalamin has been shown to significantly reduce RBC and plasma cyanide concentrations in animals and surgical patients without producing clinically important adverse effects or toxic metabolites. Thiosulfate infusions also decrease cyanide toxicity but can cause accumulation of thiocyanate resulting in clinical toxicity. Cyanocobalamin cannot effectively remove cyanide due to poor binding. CONCLUSIONS: Hydroxocobalamin is a safe and effective agent in the prevention and treatment of nitroprusside-induced cyanide toxicity. Prolonged or high-dose infusions of nitroprusside should be minimized in critically ill patients, especially if hepatic and/or renal dysfunction is present.

Animals↗

PubMedDate not supplied

Acute cyanide poisoning in prehospital care: new challenges, new tools for intervention.

Effective management of cyanide poisoning from chemical terrorism, inhalation of fire smoke, and other causes constitutes a critical challenge for the prehospital care provider. The ability to meet the challenge of managing cyanide poisoning in the prehospital setting may be enhanced by the availability of the cyanide antidote hydroxocobalamin, currently under development for potential introduction in the United States. This paper discusses the causes, recognition, and management of acute cyanide poisoning in the prehospital setting with emphasis on the emerging profile of hydroxocobalamin, an antidote that may have a risk:benefit ratio suitable for empiric, out-of-hospital treatment of the range of causes of cyanide poisoning. If introduced in the U.S., hydroxocobalamin may enhance the role of the U.S. prehospital responder in providing emergency care in a cyanide incident.

Acute Disease↗

PubMed2000

L-NAME-resistant bradykinin-induced relaxation in porcine coronary arteries is NO-dependent: effect of ACE inhibition.

1. NO synthase (NOS)inhibitors partially block bradykinin (BK)-mediated vasorelaxation. Here we investigated whether this is due to incomplete NOS inhibition and/or NO release from storage sites. We also studied the mechanism behind ACE inhibitor-mediated BK potentiation. 2. Porcine coronary arteries (PCAs) were mounted in organ baths, preconstricted, and exposed to BK or the ACE-resistant BK analogue Hyp(3)-Tyr(Me)(8)-BK (HT-BK) with or without the NOS inhibitor L-NAME (100 microM), the NO scavenger hydroxocobalamin (200 microM), the Ca(2+)-dependent K(+)-channel blockers charybdotoxin+apamin (both 100 nM), or the ACE inhibitor quinaprilat (10 microM). 3. BK and HT-BK dose-dependently relaxed preconstricted vessels (pEC(50) 8.0+/-0.1 and 8.5+/-0.2, respectively). pEC(50)'s were approximately 10 fold higher with quinaprilat, and approximately 10 fold lower with L-NAME or charybdotoxin+apamin. Complete blockade was obtained with hydroxocobalamin or L-NAME+ charybdotoxin+apamin. 4. Repeated exposure to 100 nM BK or HT-BK, to deplete NO storage sites, produced progressively smaller vasorelaxant responses. With L-NAME, the decrease in response occurred much more rapidly. L-Arginine (10 mM) reversed the effect of L-NAME. 5. Adding quinaprilat to the bath following repeated exposure (with or without L-NAME), at the time BK and HT-BK no longer induced relaxation, fully restored vasorelaxation, while quinaprilat alone had no effect. Quinaprilat also relaxed vessels that, due to pretreatment with hydroxocobalamin or L-NAME+charybdotoxin+apamin, previously had not responded to BK. 6. In conclusion, L-NAME-resistant BK-induced relaxation in PCAs depends on NO from storage sites, and is mediated via stimulation of guanylyl cyclase and/or Ca(2+)-dependent K(+)-channels. ACE inhibitors potentiate BK independent of their effect on BK metabolism.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1996

Influence of superoxide dismutase inhibition on the discrimination between NO and the nitrergic neurotransmitter in the rat gastric fundus.

1. The influence of diethyldithiocarbamate (DETCA), that irreversibly inhibits Cu/Zn-containing superoxide dismutase, on the inability of 6-anilino-5,8-quinolinedione (LY83583), hypoxanthine/xanthine oxidase, hydroquinone and hydroxocobalamin to reduce electrically-induced NANC relaxations in the rat gastric fundus was investigated. 2. Longitudinal muscle strips of the rat gastric fundus were mounted for auxotonic recording in the presence of atropine and guanethidine and tone was raised by administration of prostaglandin F2 alpha DETCA (3 x 10(-3) M) slightly reduced the short-lasting relaxations induced by 10(-5) M exogenous nitric oxide (NO) and transmural electrical stimulation for 10 s at 4 Hz but this effect was not influenced by 1000 u ml-1 superoxide dismutase (SOD). 3. DETCA (3 x 10(-5) -3 x 10(-3) M) concentration-dependently potentiated the inhibitory effect of LY83583 upon the electrically-induced relaxations, although this was less pronounced than the inhibition of the NO-induced relaxations. The inhibition of the electrically-induced non-adrenergic non-cholinergic (NANC) relaxations was not reversed by SOD while that of the NO-induced relaxations was partially reversed. 4. The inhibitory effect of hypoxanthine/xanthine oxidase, hydroquinone and hydroxocobalamin on the electrically-induced NANC relaxations in the presence of DETCA (3 x 10(-3) M) was not different from the inhibitory effect of DETCA alone. 5. It was concluded that the differentiating effect of LY83583 between exogenous NO and the endogenous nitrergic neurotransmitter is partially related to protection of the endogenous nitrergic neurotransmitter by high levels of intracellular superoxide dismutase. This mechanism does not hold for hydroquinone and hydroxocobalamin, as they still discriminate between exogenous NO and the endogenous nitrergic neurotransmitter in the presence of DETCA. The possibility that the endogenous nitrergic neurotransmitter is not free NO in the rat gastric fundus therefore remains open.

Animals↗

PubMed1971

Activities of various cobalamins for Euglena gracilis with reference to vitamin B 12 assay with Euglena.

Coenzyme B(12) and methylcobalamin in water are less active in promoting growth of Euglena gracilis Z strain than the same concentrations of cyanocobalamin and hydroxocobalamin which are equally active. When bound to human serum or human liver homogenate, however, the activities of these four cobalamins do not differ significantly with one exception. The results suggest that the Euglena assay using cyanocobalamin standards is not satisfactory for quantitation of coenzyme B(12) and methylcobalamin in water but acceptable when coenzyme B(12) and methylcobalamin are bound to serum or liver. Sulphitocobalamin in water is as active as cyanocobalamin and hydroxocobalamin but nitritocobalamin is less active. Factor B, the monocarboxylic acids of cyanocobalamin and hydroxocobalamin, and the dicarboxylic acid of cyanocobalamin in water were inactive.

Biological Assay↗

PubMed1979

Tobacco amblyopia: the effect of treatment on the electroretinogram.

In seven recently diagnosed and untreated patients with tobacco amblyopia the light and dark adapted electroretionograms were recorded together with visual acuity, central fields and the Farnsworth-Munsell 100-Hue test. After 3 months treatment with injections of hydroxocobalamin all these tests were repeated. An improvement in visual acuity resulted in all cases. A significant reduction in the Farnsworth-Munsell total error score of the seven patients was noted (p less than 0.01). A significant increase in the amplitude of the light adapted b-wave of the ERG was found (0.01 less than p less than 0.02) following the course of hydroxocobalamin injections. No other significant changes in the ERG were demonstrated. It is suggested that the ERG changes demonstrated an improvement in an abnormal biochemical state in those bipolar and perhaps Müller cell of the inner nuclear layer of the retina which make electrophysiological connnections chiefly with foveal cones.

Aged↗

PubMed1998

Remethylation defects: guidelines for clinical diagnosis and treatment.

The main remethylation defects include disorders which all have defective methionine synthesis in common. Methylenetetrahydrofolate reductase deficiency impairs methyltetrahydrofolate synthesis, defects in cytosolic reduction of hydroxocobalamin (CblC/D) impair the synthesis of both methyl- and adenosyl cobalamin and deficiencies of methionine synthase (CblE/G) are associated with defective methyl cobalamin synthesis. The clinical presentation is characterized by acute neurological distress in early infancy. In childhood, patients present with progressive encephalopathy with an end-stage which has many signs in common with the adult onset form. In fact, both have more or less severe signs of subacute degeneration of the cord. Cobalamin defective patients must be treated with parenteral supplementation of hydroxocobalamin (1-2 mg per dose). Some methylenetetrahydrofolate patients could be folate responsive and must have a high-dosage folate trial. In addition, oral betaine supplementation (2-9 g per day depending on age) appears an effective means to prevent further neurological deterioration.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1989

Vitamin B-12: placebo or neglected therapeutic tool?

Patients with normal serum vitamin B-12 levels were given injections of hydroxocobalamin (vitamin B-12b) interspersed with injections of sterile water. Those who felt better with B-12b had additional injections to establish the amount of B-12b that gave the maximum feeling of well-being. Serum vitamin B-12 tests were performed to record the level at which the patients felt best. On the same day they took a Multiphasic Personality Inventory (MMPI), an objective computerized psychological test. Hydroxocobalamin injections were then discontinued for 5 to 7 days. The patients took another serum vitamin B-12 test and another MMPI. With the higher vitamin B-12 levels, the MMPI patterns were at or closer to normal. With lower serum vitamin B-12 levels, MMPI patterns showed much more emotional distress. The author suspects that vitamin B-12 dependency disorders are common and are neglected by the medical profession.

Affect↗

PubMed1976

The forms of vitamin B12 in foods.

1. The forms of vitamin B12 were determined in foods, most of which had been prepared for consumption. 2. Five forms were detected: adenosylcobalamin, hydroxocobalamin, methylcobalamin, cyanocobalamin and sulphitocobalamin. Adenoxylcobalamin and hydroxocobalamin were the predominant forms. 3. The intestinal absorption of [57Co]sulphitocobalamin was estimated and found to be lower than that of [58Co]cyanocobalamin.

Adult↗

PubMedDate not supplied

A new spectrophotometric method for the toxicological diagnosis of cyanide poisoning.

A spectrophotometric method for the determination of hydrogen cyanide in biological fluids based on the release of cyanide ion by the addition of a strong acid and its subsequent specific reaction with hydroxocobalamin to give cyanocobalamin is proposed. The release of cyanide ion is accelerated by aeration with a stream of an inert gas (nitrogen) that carries it into the hydroxocobalamin solution. Although the in vitro reaction develops to completion within 20 min, reproducible quantitation in biological media takes 45 min. The cyanocobalamin formed is quantitated by second-derivative visible spectrophotometry from the absorbance difference between 333 and 361 nm, the measured signal being proportional to the cyanide ion concentration in the sample.

Forensic Medicine↗

PubMed2001

Negative inotropic effect of bradykinin in porcine isolated atrial trabeculae: role of nitric oxide.

OBJECTIVES: To investigate whether bradykinin affects cardiac contractility independently of its effects on coronary flow and noradrenaline release, and whether such inotropic effects, if present, are mediated via nitric oxide (NO). METHODS: Right atrial trabeculae were obtained from 35 pigs, suspended in organ baths and attached to isometric transducers. Resting tension was set at approximately 750 mg and tissues were paced at 1.5 Hz. Tissue viability was checked by constructing a concentration response curve (CRC) to noradrenaline. Next, CRCs were constructed to bradykinin, either under baseline conditions or after pre-stimulation with the positive inotropic agent forskolin (1 or 10 micromol/l), in the absence or presence of the bradykinin type 2 (B2) receptor antagonist D-Arg [Hyp3-Thi5, d-Tic7, Oic8]-bradykinin (Hoe 140) (1 micromol/l), the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) (100 micromol/l) and/or the NO scavenger hydroxocobalamin (200 micromol/l). RESULTS: Bradykinin exerted a negative inotropic effect, both with and without forskolin pre-stimulation, reducing contractility by maximally 22 +/- 3.6% (mean +/- SEM) and 23 +/- 3.6%, respectively (pEC50 8.37 +/- 0.23 and 8.62 +/- 0.22, respectively). L-NAME reduced this effect in pre-stimulated, but not in unstimulated, trabeculae. Hoe 140 and hydroxocobalamin fully blocked the inotropic effect of bradykinin. CONCLUSIONS: Bradykinin induces a modest negative inotropic effect in porcine atrial trabeculae that is mediated via B2 receptors and NO. The inconsistent results obtained with L-NAME suggest that it depends on NO synthesized de novo and/or NO from storage sites.

Animals↗