Nobuharu Yamaguchi

Regulation of norepinephrine release from cardiac sympathetic fibers in the dog by presynaptic alpha- and beta-receptors.

The effect of phenoxybenzamine (PBA), desmethylimipramine (DMI), clonidine (CLND), sotalol (STL), and isoproterenol (ISPR) on the release of endogenous norepinephrine (NE) from the heart on right cardioaccelerator nerve stimulation was studied in anesthetized dogs. Under control conditions, the catecholamine levels in coronary sinus blood increased linearly with increasing frequencies of stimulation up to 10 Hz and did not increase further at 30 Hz. The release of NE was markedly enhanced after PBA (1 mg/kg, iv) and DMI (1 mg/kg, iv). The enhanced release of NE after DMI, but not after PBA, was associated with a prolonged response in heart rate. In contrast, NE release was reduced after CLND (15 microgram/kg, iv) at stimulation frequencies of 1 and 2 Hz and this was associated with reduced responses in heart rate and left ventricular dtp/dt. STL (5 mg/kg, iv) reduced significantly the release of NE at stimulation frequencies of 1-5 Hz, whereas ISPR enhanced NE outflow at frequencies of 1-4 Hz. These results support the existence of both negative and positive feedback mechanisms on the release of norepinephrine by cardiac sympathetic fibers mediated through presynaptic alpha- and beta-adrenoreceptors, respectively. The functional significance of these mechanisms is also suggested by the correlation found between changes in NE release and variations in cardiac responses under the various drug treatements.

Correlation between the Response of the Heart to Sympathetic Stimulation and the Release of Endogenous Catecholamines into the Coronary Sinus of the Dog

The relationship between the increase in catecholamine levels of the coronary sinus b1lood and the amplitude of various cardiac responses to adrenergic nerve stimulation was studied in anesthetized dogs. Plasma catecholamine levels in both coronary sinus and aortic blood were measured by a modification of the radiometric enzymatic assay for tissue catecholamines and were found to be 0.622 ± 0.104 (SE) ng/ml and 0.933 ± 0.116 ng/ml, respectively, under basal conditions. The catecholamine levels in coronary sinus blood increased linearly during right cardioaccelerator nerve stimulation up to a frequency of 10 Hz. At this frequency, maximum values were observed in both coronary sinus blood catecholamine levels and cardiac responses. The correlation between the response in heart rate, mean coronary blood flow, and dP/dt of left ventricular pressure and the increase in endogenous catecholamine levels of coronary sinus blood was significant, but the relationship was nonlinear. The present experimental design may prove to be a reliable means of studying the role of the sympathetic nervous system in the regulation of cardiovascular function in vivo.

A new model of isolated systolic hypertension induced by chronic warfarin and vitamin K1 treatment

BACKGROUND Isolated systolic hypertension is the predominant form of hypertension in the elderly population. Reduction of arterial compliance appears to contribute to the elevation of pulse pressure (PP) and among potential mechanisms, gradual vascular calcification, fragmentation of elastic lamellae, and augmentation of rigid component like collagen could contribute to increase aortic stiffening. Few experimental models of the disease are currently available. METHODS To induce large artery calcification, rats were treated with warfarin and vitamin K(1) (WK) for 4 and 8 weeks, to inhibit the maturation of matrix Gla protein. The impact of chronic PP elevation was determined on large artery and cardiac remodeling and on aortic endothelial function. RESULTS The WK treatment led to aortic medial calcification and a proportional elevation of PP, attributable mainly to a selective elevation of systolic blood pressure. The chronic treatment also increased collagen, whereas elastin decreased in the aorta. Pulse wave velocity, an index of aortic stiffening, increased in rats treated with WK. However, indices of left ventricular and aortic hypertrophy and remodeling remained normal. In addition, the WK treatment did not modify the vasoconstriction to norepinephrine and endothelin-1, and the vasodilatory response to acetylcholine and sodium nitroprusside. CONCLUSIONS Chronic treatment with WK represents a new model of isolated systolic hypertension with several characteristics of the human disease. The relative ease to induce calcification in this model may help to foster more fundamental research, which is lacking in this type of hypertension.

Protective effects of bradykinin on the ischaemic heart: implication of the B1 receptor

1 We studied the role of bradykinin (BK) and its active metabolite Des‐Arg9‐BK on noradrenaline release in association with the incidence of ventricular arrhythmias at reperfusion of the ischaemic myocardium. 2 Experiments were performed in Langendorff perfused isolated hearts of rats subjected to 30 min no flow followed by 5 min reperfusion. The electrocardiogram was monitored continuously and noradrenaline was measured in the effluent as well as in the myocardial tissue. 3 In untreated hearts, cumulative noradrenaline overflow following global ischaemia reached 226 ± 35 pmol g−1 of heart (n = 8, P < 0.05) during the 5 min of reperfusion along with ventricular tachycardia and/or fibrillation. A decrease in myocardial noradrenaline (–31%) was also observed. 4 Bradykinin perfused at concentrations between 0.01 and 1 μm, 10 min before flow was stopped and at reperfusion, inhibited noradrenaline overflow in a concentration‐dependent manner. At a concentration of 1 μm, bradykinin completely abolished noradrenaline overflow. For the same concentration of bradykinin, myocardial noradrenaline contents were significantly higher (n = 5–8, P < 0.05). Ventricular fibrillation but not ventricular tachycardia was also prevented. 5 Des‐Arg9‐BK (0.1 μm) in the same experimental conditions had similar effects. While Hoe 140, a selective antagonist at B2 receptors, did not abolish the effects of bradykinin, Lys [Leu8] Des‐Arg9‐BK, an antagonist at B1 receptors, abolished the effects of both Des‐Arg9‐BK and bradykinin. 6 These results suggest that the cardioprotective action of bradykinin in the preparation may be mediated partially by an inhibitory effect on noradrenaline liberation which could be mediated by the activation of B1 receptors.

Basal somatodendritic dopamine release requires snare proteins.

Dopaminergic neurons have the capacity to release dopamine not only from their axon terminals, but also from their somatodendritic compartment. The actual mechanism of somatodendritic dopamine release has remained controversial. Here we established for the first time a rat primary neuron culture model to investigate this phenomenon and use it to study the mechanism under conditions of non‐stimulated spontaneous firing (1–2 Hz). We found that we can selectively measure somatodendritic dopamine release by lowering extracellular calcium to 0.5 mm, thus confirming the previously established differential calcium sensitivity of somatodendritic and terminal release. Dopamine release measured under these conditions was dependent on firing activity and independent of reverse transport through the plasma membrane. We found that treatment with botulinum neurotoxins A and B strongly reduced somatodendritic dopamine release, thus demonstrating the requirement for SNARE proteins SNAP‐25 and synaptobrevin. Our work is the first to provide such direct and unambiguous evidence for the involvement of an exocytotic mechanism in basal spontaneous somatodendritic dopamine release.

Transient involvement of endothelin in hypertrophic remodeling of small arteries.

Objective: This study was designed to evaluate the capacity of norepinephrine (NE) to induce hypertrophic remodeling of small arteries in rats, and to determine the involvement of endothelin (ET) to initiate and maintain it. Design and results: Treatment with NE (2.5 μ g/kg per min) for 14 or 28 days produced a similar inward hypertrophic remodeling, characterized by a smaller lumen, but increased media thickness and cross-sectional area. Arterial stiffness was reduced. Histological evaluation confirmed the hypertrophic nature of remodeling. Concomitant administration of LU135252 (ET-receptor antagonist) for the first 14 days of NE administration prevented the development of hypertrophy, without altering arterial mechanics. Treatment with the same antagonist from day 14 to day 28 of NE or angiotensin II (Ang II) treatment failed to regress established vascular hypertrophy. In contrast, normalization of arterial structure was observed with prazosin, an α-adrenergic blocker. Endothelin content in small mesenteric arteries showed a transient elevation following chronic NE administration. Conclusions: Increased circulating NE levels are associated with hypertrophic remodeling of small arteries, in which ET plays an initiating role. However, the maintenance of vascular hypertrophy is ET-independent, either in the presence of augmented circulating levels of NE or Ang II. Thus, early rather than late treatment with ET-receptor antagonists may be a preferable approach to limit small artery-mediated end-organ damage in cardiovascular diseases.

Role of L-type Ca2+ channel in PACAP-induced adrenal catecholamine release in vivo

The aim of the present study was to investigate whether the dihydropyridine-sensitive L-type Ca2+ channel is operative in adrenal catecholamine (CA) secretion induced by a novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were determined by a high-performance liquid chromatography method. All drugs tested were locally infused into the left adrenal gland via the left adrenolumbar artery. PACAP, with the isoform consisting of 27 (PACAP-27) and 38 (PACAP-38) amino acid residues, significantly increased CA output in a dose-dependent manner, with doses ranging from 5 to 500 ng and 7 to 700 ng, respectively. However, the amplitude of epinephrine response to PACAP-27 was three times greater than that obtained with PACAP-38 at the highest dose tested. In a separate group, a single dose of PACAP-27 (50 ng) induced highly reproducible CA responses when the same dose was repeated with an interval of 35 min. In dogs treated with nifedipine (50 microg), 5 min before the second administration of PACAP-27, the net CA response was significantly inhibited by approximately 50% compared with that obtained in the presence of vehicle. A similar CA response to BAY K 8644 (5 microg) was completely abolished by the same dose of nifedipine. The present results indicate that both PACAP-27 and PACAP-38 have the direct local secretagogue effect on the adrenal medulla in vivo and that CA responses to PACAP-27 were greater than those observed with PACAP-38 at equivalent mole doses. The study suggests that the dihydropyridine-sensitive L-type Ca2+ channel is functionally involved in PACAP-induced adrenal CA secretion in the canine adrenal medulla in vivo.The aim of the present study was to investigate whether the dihydropyridine-sensitive L-type Ca2+ channel is operative in adrenal catecholamine (CA) secretion induced by a novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were determined by a high-performance liquid chromatography method. All drugs tested were locally infused into the left adrenal gland via the left adrenolumbar artery. PACAP, with the isoform consisting of 27 (PACAP-27) and 38 (PACAP-38) amino acid residues, significantly increased CA output in a dose-dependent manner, with doses ranging from 5 to 500 ng and 7 to 700 ng, respectively. However, the amplitude of epinephrine response to PACAP-27 was three times greater than that obtained with PACAP-38 at the highest dose tested. In a separate group, a single dose of PACAP-27 (50 ng) induced highly reproducible CA responses when the same dose was repeated with an interval of 35 min. In dogs treated with nifedipine (50 μg), 5 min before the second administration of PACAP-27, the net CA response was significantly inhibited by ∼50% compared with that obtained in the presence of vehicle. A similar CA response to BAY K 8644 (5 μg) was completely abolished by the same dose of nifedipine. The present results indicate that both PACAP-27 and PACAP-38 have the direct local secretagogue effect on the adrenal medulla in vivo and that CA responses to PACAP-27 were greater than those observed with PACAP-38 at equivalent mole doses. The study suggests that the dihydropyridine-sensitive L-type Ca2+ channel is functionally involved in PACAP-induced adrenal CA secretion in the canine adrenal medulla in vivo.

5-Hydroxytryptamine is biotransformed by CYP2C9, 2C19 and 2B6 to hydroxylamine, which is converted into nitric oxide.

There is circumstantial evidence suggesting that 5‐hydroxytryptamine (5‐HT) could be biotransformed by enzymatic systems other than monoamino oxidase A, and that the isoforms of cytochrome P450 may be a source of nitric oxide. This study aimed to assess whether cytochrome P450 contributes to 5‐HT biotransformation, and to provide evidence that 5‐HT metabolism generates nitric oxide. Addition of 5‐HT to cultured hepatocytes yielded 5‐hydroxyindol acetic acid, a formation modulated by cytochrome P450 enzyme inducers and inhibitors. Recombinant human CYP2B6, 2C9 and 2C19 biotransformed 5‐HT in 5‐hydroxyindol acetic acid, but not CYP1A2, 2D6 or 3A4. Cultured hepatocytes with 5‐HT generated nitric oxide, the amount of which was altered by cytochrome P450 enzyme inducers and inhibitors. In the presence of CYP2B6, 2C9 and 2C19, 5‐HT relaxed precontracted isolated aortic rings, with or without endothelium, an effect prevented by the addition of methylene blue and an inhibitor of catalase, but not by myoglobin. In the absence of catalase, hydroxylamine was always assayed as a byproduct of 5‐HT metabolism. In conclusion, CYP2B6, 2C9 and 2C19 biotransform 5‐HT, yielding hydroxylamine, which is converted to nitric oxide in the presence of catalase.

Myocardial dysfunction and norepinephrine release in the isolated rat heart injured by electrolysis-induced oxygen free radicals

In the present investigation, we used electrolysis as a source of oxygen free radicals to test their possible role in norepinephrine release, as well as in the mechanism of cellular injury, cardiac dysfunction and arrhythmias. In the isolated rat heart perfused under constant pressure, according to the Langendorff technique, electrolysis of the Krebs-Henseleit solution (10 mA d.c. current for 1 min) produced myocardial irreversible dysfunction within 5 min. Fifteen minutes after electrolysis, significant falls in the left ventricular pressure (from 87.5 +/- 6.8 to 33.7 +/- 5.2 mmHg), dP/dt max (from 1230 +/- 90 to 375 +/- 59 mmHg/s), heart rate (from 287 +/- 18 to 119 +/- 13.5 beats/min) and coronary flow (from 14.8 +/- 9 to 3.4 +/- 1.7 ml/min) were observed, along with an increase in left ventricular end diastolic pressure from 10 to 50 +/- 3.5 mmHg (n = 8, P less than 0.01). AV conduction block and/or sinus bradycardia were noted in all preparations. An increase in norepinephrine washout from 298.5 +/- 84 at baseline to 610 +/- 110 pg/min/g 5 min after electrolysis was measured (n = 8, P less than 0.05) and a 44.8 +/- 9.2% and 35 +/- 7.5% reduction, respectively in right and left ventricular tissue norepinephrine content was also found at 30 min (n = 5, P less than 0.05). Pretreatment of the hearts 10 min before electrolysis and throughout the experimental period by superoxide dismutase (SOD; 100 U/ml), catalase (150 U/ml), a combination of SOD + catalase or mannitol (50 mM) partially blocked the deleterious effect of free radicals and permitted a functional recovery of 50 to 60%, mannitol being the more potent protective agent. Furthermore, these scavengers also significantly reduced norepinephrine washout.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of adrenal catecholamine release by PACAP in vivo

The aim of the present study was to investigate whether pituitary adenylate cyclase-activating polypeptide-(1-27) (PACAP27) can modulate the adrenal catecholamine (CA) secretion induced by splanchnic nerve stimulation (SNS) and by exogenous acetylcholine (ACh) in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were quantified by a high-performance liquid chromatography coupled with electrochemical detection. Adrenal venous blood flow was measured by gravimetry. Local infusion of PACAP27 (0.5, 5, and 50 ng) to the left adrenal gland via the adrenolumbar artery resulted in an increase in CA output, reaching a significant level at the highest dose tested. Either direct SNS (2 Hz) or local infusion of ACh (0.5 μg) to the left adrenal gland produced significant increases in CA output to an extent similar to that obtained with 50 ng of PACAP27 alone. In the presence of PACAP27 (50 ng), CA responses to either SNS or exogenous ACh were significantly potentiated by approximately four- and sixfold, respectively, compared with those obtained in response to each stimulus alone. However, the enhanced CA responses to ACh were not significantly different from those to SNS. The results indicate that the increase in adrenal CA secretion, induced by either direct SNS or exogenous ACh, is synergistically enhanced by PACAP27. The study suggests that the enhanced CA secretion may result from the activation of a PACAP-mediated facilitatory mechanism(s) localized presumably at the postsynaptic level in the canine adrenal medulla in vivo, although the possible involvement of presynaptic mechanisms cannot completely be ruled out in the present study.