Takaharu Ishibashi

Relationship between myoglobin contents and increases in cyclic GMP produced by glyceryl trinitrate and nitric oxide in rabbit aorta, right atrium and papillary muscle

SummaryEffects of glyceryl trinitrate (GTN) and nitric oxide (NO) on the cardiac functions and myocardial cyclic GMP (cGMP) contents were examined in comparison with those in the aorta and correlated with myoglobin (an inhibitor of soluble guanylate cyclase) contents using the preparations isolated from the reserpinized rabbit.GTN (10−10-10−4mol/l) produced a dose-dependent relaxation in the aorta. However, this compound exerted no effect on the rate of the spontaneous beat of the right atrium and the contraction of the papillary muscle. A transient and significant increase in cGMP was observed in the aorta with GTN (3 × 10−6 mol/l). Although the increase was also observed in the right atrium, it was much smaller. No definite change was observed in papillary muscle. Increases in cGMP produced by NO (3 × 10−6 mol/l) were larger and significant in all tissues; (AUCcGMP(GTN)/AUCcGMP(NO)) ratio was 30.1 for the aorta, 65.0 for the right atrium and 16.3% for the papillary muscle. Although higher concentrations of NO were necessary in the right atrium and papillary muscle to induce increases in cGMP, no differences were noted in the three tissues as regards the maximum accumulation of this substance. Furthermore, kinetic analysis of NO-induced increases in tissue cGMP indicated no marked difference in the production rate among the three tissues, while the rate of elimination of cGMP was lower in the aorta than in the atrium or the papillary muscle. The increases in cGMP observed in these three tissues were inversely related to the contents of myoglobin in respective tissues. No effect on myocardial function was observed with NO up to the concentration of 3 × 10−5 mol/l.These results suggest that myoglobin, an endogenous inhibitor of activation of soluble guanylate cyclase by NO, was responsible for the lower production of cGMP by NO and GTN in the myocardial tissue.

Vasodilating properties of KRN2391: structural basis of a new pyridine-type potassium channel opener with a nitrate moiety

SummaryThe vasodilating mechanism of a new compound, cyanoimino-3-pyridylmethylaminoethyl nitrate methanesulfonate (KRN2391), a derivative of nicorandil, was examined in the isolated rabbit aorta. To elucidate the structure activity relationship, a comparison was made with the two denitrated derivatives: cyanoimino-3-pyridylmethylaminoethyl acetate methanesulfonate (Ki4032) and cyanoimino-3-pyridylmethylaminoethyl alcohol (Ki3315).In preparations precontracted with phenylephrine (10−7 mol/l), KRN2391, Ki4032 and Ki3315 caused concentration-dependent relaxation. pD2 values (−log [EC50]) were 6.74 ± 0.03, 5.67 ± 0.05 and 3.63 ± 0.03, respectively. Both methylene blue and glibenclamide produced a shift to the right of the concentration-response curves for KRN2391. The shift by glibenclamide became greater in the presence of methylene blue. An elevation of the cGMP content was not detected until the concentration of KRN2391 was increased to a level enough to produce a full relaxation (3 × 10−6 mol/l). In contrast, in the case of Ki3315 a parallel shift to the right of the concentration-response curve was observed after glibenclamide (10−5 mol/l). Methylene blue (10−5 mol/l) had no effect on the concentration-response curve, and there was no increase in cyclic GMP (cGMP) with 10−5 mol/l of the compound. The concentration-response curve of Ki4032 was also attenuated by glibenclamide. Though this compound lacks the nitrate moiety, it (10−4 mol/l) showed a slight tendency to increase the cGMP content, and methylene blue slightly but significantly modified the concentration-response curve of this compound. However, the co-administration of glibenclamide and methylene blue resulted in no further modification of the concentration-relaxation curve.These results indicate that both the opening of potassium channels and the activation of the soluble guanylate cyclase are responsible for the vasorelaxant effect of KRN2391. These two mechanisms are operative at similar concentrations of KRN2391. In contrast, Ki3315 and Ki4032 are exclusive potassium channel openers. Although KiA032 produced a slight increase in cGMP, this is probably not due to the activation of soluble guanylate cyclase.

Na+/H+ exchange is not operative under low-flow ischemic conditions

Using 31P-NMR the existence of Na+/H+ exchange system and its contribution to intracellular pH (pHi) regulation were examined in the isolated isovolumic rat heart under physiological and pathophysiological conditions. Ethylisopropylamiloride (EIPA) was used as a tool to search into the role of Na+/H+ exchange system. In the normal well-oxygenated heart dose-dependent negative chronotropic effects were observed with 10(-6) to 10(-5) M EIPA. After 10(-4) M the heart ceased to beat and a progressive fall of high energy phosphates compounds occurred. However, contrary to expectation pHi did not fall but rose after EIPA. In NH4Cl-loaded hearts removal of NH4Cl resulted in a fall of the pHi followed by a rapid recovery to the normal pHi. After 10(-5) M EIPA the fall of pHi became greater and there was no recovery within 35 min of observation period. This dose of EIPA, however, did not affect the time course of changes in the pHi during 60 min of low-flow ischemia (0.2 ml/min). It is concluded that pHi regulation following an acute acid loading is dependent on amiloride-sensitive Na+/H+ exchange. However, Na+/H+ exchange system does not play an important role in maintenance of the pHi under normoxic or ischemic condition. In the normoxic heart EIPA produced a decrease in heart rate without producing any change either in myocardial energy metabolism or in pHi. Thus, the compound could be categorized as a bradycardic agent.

2-Nicotinamidoethyl acetate (SG-209) is a potassium channel opener: Structure activity relationship among nicorandil derivatives

SummaryThe mechanism of the vasodilating action of 2-nicotinamidoethyl acetate (SG-209), a derivative of nicorandil, was examined in the isolated rabbit aorta. Comparison was made using 2-nicotinamidoethyl alcohol (SG-86) and 2-nicotinamidoethyl nitrate (nicorandil; SG-75) to reveal any structure-activity relationships.SG-209 and nicorandil caused concentration-dependent relaxation in preparations precontracted with phenylephrine (10−7 mol/l), while SG-86 produced a relaxation only at very high concentrations. The pD2 values (−log[EC50]) of SG-209 and nicorandil were 3.59 ± 0.07 and 5.95 ± 0.10, respectively. The vasorelaxant activity of nicorandil was associated with significant increases in cyclic GMP content, while that of SG-209 was not. Methylene blue (10−5 mol/l) attenuated the relaxant effect of nicorandil, but had no effect on that of SG-209. Furthermore, the relaxant effect of nicorandil was not affected by glibenclamide (10−5 mol/l), whilst the relaxant effect of SG-209 was abolished by this compound. In the presence of methylene blue (10−5 mol/l), however, glibenclamide (10−5 mol/l) attenuated the relaxant effect of higher concentrations of nicorandil (≥ 10−5 mol/l).These results indicate that the relaxant effect of SG-209 is mostly if not exclusively due to the activation of potassium channels, while this action contributes to the vasodilating action of nicorandil only at higher concentrations.

Effects of sodium nitroprusside (MR7S1) and nitroglycerin on the systemic, renal, cerebral, and coronary circulation of dogs anesthetized with enflurane

SummaryIn beagle dogs anesthetized with enfluranenitrous oxide, effects of sodium nitroprusside (SNP; MR7S1) and nitroglycerin (NTG) on hemodynamics and main organ circulation were studied to evaluate their effectiveness and safety as hypotensive agents during anesthesia. SNP (MR7S1) infusion (1–10 μg/kg/min) decreased arterial blood pressure in a dose-dependent manner. The hypotension was stable during the infusion. After discontinuation of infusion, the blood pressure rapidly returned to the initial level. The hypotension was associated with decreases in cardiac output and total peripheral resistance. NTG infusion (3–10 μg/kg/min) decreased arterial blood pressure, too, but the hypotension was less marked and not dose dependent, and the recovery was slower. Neither drug changed the heart rate. Infusion of SNP (MR7S1) and NTG did not change the hypotension induced by the injection of adenosine, SNP, and NTG. Furthermore, cerebral blood flow, cerebral oxygen consumption, and renal blood flow were unchanged during the hypotension produced by either drug. Coronary blood flow was decreased, but this was due to decreases in cardiac oxygen consumption. In conclusion, SNP (MR7S1) is superior to NTG as a hypotensive agent during anesthesia in efficacy, clear dose dependency, and rapid recovery. The hypotension induced by NTG as well as SNP (MR7S1) seems to have no undesirable effects on the circulation of important organs.

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR.

Using31P-,23Na- and39K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na+, a shift reagent (Dy(TTHA)3−) was used in23Na-NMR study. In39K-NMR study, the extracellular K+ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K+ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na+ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na+−H+ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na+ and a decrease in intracellular K+ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na+ and associated decrease in intracellular K+ may be explained by inhibition of the Na+−K+ ATPase due to the depletion of ATP. A longer observation with31P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.

Hemodynamic effects of benazepril, an angiotensin-converting enzyme inhibitor, as studied in conscious normotensive dogs

SummaryHemodynamic effects and inhibitory effects on the pressor response to exogenous angiotensin I of benazepril (CGS 14824A), a new angiotensin-converting enzyme (ACE) inhibitor, were examined in conscious chronically instrumented normotensive dogs in comparison with those of captorpil. Oral administration of benazepril (1–10 mg/kg) and captopril (3 and 10 mg/kg) reduced the blood pressure and inhibited the pressor response to angiotensin I dose-dependently. The blood-pressure-lowering effect of benazepril was as potent as that of captopril. The onset of effects of benazepril was slower and the duration longer than that of captopril. There was no close correlation between the attenuation of pressor response to exogenous angiotensin I and the blood-pressure-lowering effect of these two agents. These results indicate that benazepril is a potent ACE inhibitor with a slow onset and a long duration. The slow onset of action may be explained by the necessity of prior conversion of this compound to an active metabolite. A mechanism or mechanisms other than that responsible for the inhibition of pressor response to exogenous angiotension I must be taken into consideration to explain the blood-pressure-lowering effects of benazepril and captopril.

IP3 production in A10 cells, an established aortic smooth muscle cultured cell line: effects of agonist administration procedure and culture conditions.

1. After normal culture (10% FBS), medium exchange itself evoked marked increases in IP3 levels in A10 cells that masked the net change in IP3 in response to vasopressin (100 nM). 2. Low-serum (0.3% FBS) culture for 10 days abolished the increase in IP3 by medium exchange, and increased expression of cGMP kinase. 3. Stimulation of cells with vasopressin (100 nM) by a pipetting procedure, increased IP3 levels irrespective of culture conditions. 4. This cell line therefore could be used in a contractile form (after low-serum culture) with pipetting procedure to observe IP3 response, especially with regard to interactions with the cGMP signaling system.

Relationship between coronary flow and adenosine release during severe and mild hypoxia in the isolated perfused rat heart with special reference to time-course change

SummaryThe contribution of endogenous adenosine to coronary vasodilation induced by global myocardial hypoxia was examined. In isolated rat hearts perfused by means of Langendorffs technique, the relationship between chronological changes in coronary flow and adenosine release during hypoxia was analyzed. The oxygenation level of myoglobin (MbO2), myocardial oxygen uptake, lactate release, and left ventricular pressure (LVP) was also measured. Adenosine was determined by radio-immunoassay, and the MbO2 levels by the optical method. Severe hypoxia (20% O2+75% N2+5% CO2) increased coronary flow, adenosine release, and lactate release and decreased both myocardial oxygen uptake and LVP. Mild hypoxia (50% O2+45%N2+5%CO2) also increased coronary flow, adenosine release, and lactate release, while it affected neither myocardial oxygen uptake nor LVP. These results suggest that the oxygen supply is compensated by an increase in coronary flow in mild hypoxia, whereas this does not occur in severe hypoxia. Changes in MbO2 were the reverse of those in coronary flow during severe hypoxia, confirming that a decrease in intracellular oxygen correlates well with an increase in coronary flow. The pattern of changes in adenosine release, however, was not identical with that in coronary flow in severe and mild hypoxia, indicating that there is no significant relationship between coronary flow and adenosine release in either severe or mild hypoxic hearts. These findings suggest that adenosine is not the only metabolic mediator of regulation of coronary flow in hypoxic hearts.