Toji Yamazaki

Myocardial interstitial norepinephrine and dihydroxyphenylglycol levels during ischemia and reperfusion

OBJECTIVE The aim was to elucidate the relation between norepinephrine (NE) release and intraneuronal NE kinetics in the ischemic region of the in vivo heart. METHODS Using dialysis technique in the heart of anesthetized cats, we sampled dialysate from the ischemic region during 120-min coronary occlusion and reperfusion. Dialysate NE and dihydroxyphenylglycol (DHPG) contents were measured as indexes of myocardial interstitial NE and DHPG levels. RESULTS Within 20 min of occlusion, interstitial NE levels increased while DHPG levels decreased. This NE increase was suppressed by omega-conotoxin GVIA and enhanced by desipramine. These data suggest that axoplasmic NE levels increased by neuronal reuptake following exocytotic release, while intraneuronal DHPG production was suppressed due to the reduced monoamine oxidase activity. After 20 min of occlusion, interstitial NE levels increased markedly, accompanied by increased DHPG levels. This NE increase was suppressed by desipramine. These findings imply that NE mobilization from stored vesicles to axoplasma exceeded outward NE transport through uptake(1) carrier in the amount of NE, and that a substantial increase in axoplasmic NE levels compensated for the reduced monoamine oxidase activity. After reperfusion, interstitial NE levels rapidly decreased while DHPG levels increased further. Both responses in NE and DHPG were suppressed by desipramine, indicating the involvement of recovered neuronal reuptake function. CONCLUSIONS In the ischemic region, intraneuronal DHPG production was affected by alterations in monoamine oxidase activity, NE mobilization from stored vesicles, and carrier-mediated NE transport. The involvement of these factors in intraneuronal NE kinetics varied with the time of occlusion and reperfusion.

Routine high-performance liquid chromatographic determination of myocardial interstitial norepinephrine

The present study describes a high-performance liquid chromatographic-electrochemical detection (HPLC-ED) system for routine measurement of the low levels of norepinephrine (NE) found in the myocardial interstitial space. In this system, an in vivo detection limit of 100 fg in a 50-microliters injection was achieved for NE. Using cardiac dialysis technique, 20-microliters dialysates were sampled from the myocardial interstitial space at 2-min intervals. The basal dialysate NE concentration was 16.6 +/- 4.0 pg/ml. This low detection limit allowed the dialysate NE concentration to be monitored for dysfunction of the cardiac sympathetic nerve terminal. This system offers a new possibility for routine analysis of myocardial interstitial NE levels.

In vivo direct monitoring of vagal acetylcholine release to the sinoatrial node

To directly monitor vagal acetylcholine (ACh) release into the sinoatrial node, which regulates heart rate, we implanted a microdialysis probe in the right atrium near the sinoatrial node and in the right ventricle of anesthetized rabbits, and perfused with Ringers solution containing eserine. (1) Electrical stimulation of right or left cervical vagal nerve decreased atrial rate and increased dialysate ACh concentration in the right atrium in a frequency-dependent manner. Compared to left vagal stimulation, right vagal nerve stimulation decreased atrial rate to a greater extent at all frequencies, and increased dialysate ACh concentration to a greater extent at 10 and 20 Hz. However, dialysate ACh concentration in the right atrium correlated well with atrial rate independent of whether electrical stimulation was applied to the right or left vagal nerve (atrial rate=304-131 x log[ACh], R(2)=0.77). (2) Right or left vagal nerve stimulation at 20 Hz decreased atrial rate and increased dialysate ACh concentrations in both the right atrium (right, 17.9+/-4.0 nM; left, 7.9+/-1.4 nM) and right ventricle (right, 0.9+/-0.3 nM; left, 1.0+/-0.4 nM). However, atrial dialysate ACh concentrations were significantly higher than ventricular concentrations, while ventricular dialysate ACh concentrations were not significantly different between right and left vagal nerve stimulation. (3) The response of ACh release to right and left vagal nerve stimulation was abolished by intravenous administration of a ganglionic blocker, hexamethonium bromide. In conclusion, ACh concentration in dialysate from the right atrium, sampled by microdialysis, is a good marker of ACh release from postganglionic vagal nerves to the sinoatrial node.

Liquid chromatographic determination of myocardial interstitial epinephrine

This study describes a high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) for monitoring of epinephrine (Epi) in the myocardial interstitial space. The in vitro detection limit for Epi was 200 fg in a 50-microl injection. Using a cardiac dialysis technique, 60-microl dialysates were sampled from the myocardial interstitial space (6-min fractions). After an alumina procedure, the dialysate Epi concentration was measured using the HPLC-ED system. Although the basal Epi concentration was undetectable, local administration of desipramine increased Epi concentration of the dialysate to 38.1+/-18.5 pg/ml. This system affords a new possibility for estimating myocardial interstitial Epi level.

Vagal stimulation suppresses ischemia-induced myocardial interstitial myoglobin release

AIMS To evaluate vagal stimulation-mediated myocardial protection against ischemia and reperfusion in in vivo ischemic myocardium. MAIN METHODS We measured myocardial interstitial myoglobin levels in the ischemic region using a cardiac microdialysis technique in anesthetized and vagotomized cats. We occluded the left anterior descending coronary artery (LAD) for 60 min and reperfused it for 60 min (VX group, n = 6). The effects of bilateral vagal stimulation (10 V, 5 Hz, 1-ms pulse duration), initiated immediately after LAD occlusion, were examined (VS group, n = 6). To examine the involvement of phosphatidylinositol 3-kinase (PI3K), vagal stimulation was performed after pretreatment with a PI3K inhibitor wortmannin (0.6 mg/kg, i.v.) (VS-W group, n = 6). To examine the contribution of bradycardia, vagal stimulation was performed with fixed-rate ventricular pacing (VS-P group, n = 6). KEY FINDINGS The average myoglobin level during the ischemic period was 1170+/-141 in VX (in ng/ml, mean+/-SE), which was significantly attenuated in VS (466+/-87, P<0.05) and VS-W (613+/-124, P<0.05) but not in VS-P (953+/-203). Reperfusion increased the myoglobin level to 2500+/-544 in VX, whereas it was suppressed in VS (824+/-213, P<0.05) and VS-W (948+/-315, P<0.05) but not in VS-P (1710+/-253). SIGNIFICANCE Vagal stimulation, initiated immediately after LAD occlusion, attenuated the myocardial injury. Moreover, bradycardia, independent of PI3K pathway, plays a significant role in vagally induced cardioprotection during acute myocardial ischemia.

Effects of ouabain on in situ cardiac sympathetic nerve endings

Using dialysis technique, the effects of ouabain on in situ cardiac sympathetic nerve endings were examined in anesthetized cats. Dialysis probes were implanted in the left ventricular myocardium, and the concentration of dialysate norepinephrine (NE) was used as an indicator of NE output at the cardiac sympathetic nerve ending. Locally applied ouabain dose-dependently (1, 10, 100 microM) increased dialysate NE levels. This finding suggested that ouabain causes an increase in NE efflux without any requirement for prior mobilization of NE from vesicular stores. Transection of sympathetic nerves innervating the heart, was without effect on the ouabain (100 microM)-induced increase in NE efflux. Pretreatment with a Ca2+-channel blocker, omega-conotoxin GVIA (10 microg/kg i.v.) suppressed the ouabain-induced NE efflux. These data suggested that ouabain opened N-type calcium channels coupled to NE release without centrally mediated neural transmission. Furthermore, ouabain-induced NE efflux was suppressed by pretreatment with desipramine (neuronal NE uptake inhibitor, 100 microM). Our data suggest that the two mechanisms (exocytosis and carrier-mediated outward transport), to the same extent, contributed to the amount of NE efflux evoked by ouabain in in situ cardiac sympathetic nerve endings.

Effects of locally administered desipramine on myocardial interstitial norepinephrine levels.

We investigated whether locally administered neuronal uptake blockade with desipramine (DMI) modified basal myocardial interstitial norepinephrine (NE) levels and stimulation-induced NE responses. Using the dialysis technique, dialysates were sampled from the midwall of the left ventricle in anesthetized cats. Dialysate NE concentrations, as an index of myocardial interstitial NE level, were measured by high performance liquid chromatography. Through the dialysis probe, local administration of DMI (10(-4) mol/l) increased the dialysate NE level from 24.6 +/- 12.2 to 127.8 +/- 37.4 and from 11.9 +/- 5.4 to 64.9 +/- 28.7 pg/ml regardless of whether stellate ganglia were intact or transected. The arterial pressure and heart rate responses to electrical stimulation of the stellate ganglia were not altered by DMI. Control nerve stimulation increased dialysate NE concentrations to 214.5 +/- 100.0 and 57.5 +/- 22.0 pg/ml at 5 Hz and 2 Hz, respectively. During local administration of DMI, nerve stimulation-induced NE response increased to 997.5 +/- 202.0 at 5 Hz and to 436.0 +/- 68.5 pg/ml at 2 Hz. We conclude that local administration of DMI caused marked rises in basal myocardial interstitial NE concentrations and responses to cardiac sympathetic activation.

Effects of brief ischaemia on myocardial acetylcholine and noradrenaline levels in anaesthetized cats

Although brief ischaemic events make the myocardium tolerant to subsequent prolonged ischaemia, known as ischaemic preconditioning, whether brief ischaemia also affects neural regulation at the in vivo heart remains unknown. We examined the effects of brief ischaemia on myocardial interstitial acetylcholine (ACh) and noradrenaline (NA) levels in anaesthetized cats (n = 6). Baseline ACh and NA levels were 0.65 +/- 0.13 and 0.66 +/- 0.17 nmol l(-1) (mean +/- SE), respectively. Two sets of 5-min brief occlusion followed by 20-min reperfusion of the left anterior descending coronary artery (LAD) significantly increased the myocardial interstitial ACh level to 4.6 +/- 0.7 nmol l(-1) (P < 0.01), while not affecting the myocardial interstitial NA level. Subsequent 60-min LAD occlusion significantly increased the ACh and NA levels to 34.9 +/- 6.0 and 96.5 +/- 17.0 nmol l(-1) (P < 0.01), respectively. Vagotomy abolished the myocardial interstitial ACh release during brief ischaemia and attenuated the ACh release during subsequent 60-min ischaemia (n = 6). In contrast, vagotomy did not affect the subsequent ischaemia-induced myocardial interstitial NA release. We conclude that the brief ischaemia affects myocardial interstitial ACh release but not NA release in the ischaemic myocardium in vivo.

Effects of ω-conotoxin GVIA on cardiac sympathetic nerve function

Abstract Using a cardiac dialysis technique, the effects of ω -conotoxin GVIA ( N -type Ca 2+ channel blocker) on cardiac sympathetic nerve function was examined in anesthetized cats. Dialysis probes were implanted in the left ventricular wall and the concentration of dialysate norepinephrine (NE) served as an indicator of NE output at cardiac sympathetic nerve endings. Administration of ω -conotoxin GVIA (10 μ g/kg i.v.) suppressed dialysate NE responses to the nerve stimulation. The ouabain (1 μ M) induced NE increment was less markedly suppressed by ω -conotoxin GVIA. Furthermore, ω -conotoxin GVIA neither influenced neuronal NE uptake nor tyramine induced release of NE from stores. These findings suggest that the neuronal effect of ω -conotoxin GVIA is attributable to a reduction of exocytotic NE release without alterations of neuronal NE uptake or storage. Cardiac dialysis with ω -conotoxin GVIA offers a new approach for the discrimination between Ca 2+ dependent exocytotic and non-exocytotic NE release.

Norepinephrine efflux evoked by potassium chloride in cat sympathetic nerves: dual mechanism of action

Using a dialysis technique, prominent efflux of norepinephrine (NE) from cardiac sympathetic nerve endings was observed under local administration of potassium chloride (KCl, 100 mM). KCl induced NE efflux was suppressed by omega-conotoxin GVIA or desipramine but residual efflux of NE was still detectable. In the presence of omega-conotoxin GVIA, KCl induced efflux of NE was augmented by pretreatment with reserpine, indicating that this efflux of NE was derived from axoplasma with neurotransporter. These data suggest that a KCl induced brisk increase in dialysate NE levels might occur as a consequence of exocytotic NE release and carrier mediated outward NE transport from nerve endings.