Effects Of Sympathetic Histamine On Vasomotor Responses Of Blood Vessels In Rabbit Ears

Our previous studies showed that histamine has a wide distribution in the sympathetic nervous systems of different species, including the mouse, guinea pig, dog, monkey, etc., and that stimulation of the sympathetic nerve terminals can simultaneously trigger the release of histamine (HA) and norepinephrine (NE). These results indicate that histamine is a very likely neurotransmitter which coexists with NE in sympathetic nerves.Most blood vessels are innervated by sympathetic nerves. Since histamine is found in and can be released from sympathetic nerves, we wonder whether this neurogenic histamine can mediate some pre- or post-synaptic effects in blood vessels. To answer this question, isolated perfused rabbit ears were used and the changes in the condition of vasoconstrictor induced by electrical stimulation (ES) were observed after administration of different histamine receptor antagonists; and a modified method of high performance liquid chromatogram (HPLC) with electrochemical detector (ECD) was developed for determination of histamine and NE in the perfusate to certify the release of sympathetic histamine. The purpose of this study is to explore the effects of sympathetic histamine on vasomotor responses and to provide further evidence to reveal the transmitter character of sympathetic histamine.1 A modified method for simultaneous determination of histamine and norepinephrine using high performance liquid chromatography with electrochemical detectionTo detect the content of histamine and NE in perfusate simultaneously, a modified method of HPLC-ECD was established to overcome some drawbacks, such as complicated procedures and relatively low sensitivity, of the literature method. The mixed solution of O-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) in the proper proportion was used as derivative reagent to precolumn derivatize histamine and NE. 3-methylhistamine (3-M-His) and 3,4-dihydroxybenzylamine (DHBA) were used as internal standards of histamine and NE, respectively. Meanwhile, the effects of conditions of derivative reaction and HPLC operation on response values were also investigated. Conditions took into consideration included reaction pH, reaction time, the proportion of 2-ME in derivative reagent and the operating voltage. The regression equation of working curve for HA was Y = 0.868 X - 0.1233 with linear range of 0.25~10 ng/ml, correlation coefficientγ= 0.9992, 3-M-His 100 ng/ml. The regression equation of working curve for NE was Y = 0.0832 X - 0.1601 with linear range of 2.5~100 ng/ml, correlation coefficientγ= 0.9995, DHBA 500 ng/ml. The detection limit for HA and NE was 0.125 ng/ml and 1.25 ng/ml, respectively. The method is easy to operate with high sensibility. 2 Effects of sympathetic histamine on vasomotor responses of blood vessels in rabbit ear to electrical stimulationIsolated perfused rabbit whole ears were used to investigate the effects of sympathetic histamine release induced by different frequencies of ES on vasomotor responses. ES was applied to posterior auricular nerves of the whole rabbit ear at 10 Hz, 20 Hz and 40 Hz, respectively. Besides, the whole ear was perfused with different histamine receptor antagonists under constant perfusion pressure, and the changes in the flow rate of perfusate were observed. The flow rate of perfusate can represent the vasomotor condition to a certain extent. The flow rate of venous outflow was decreased by ES at all the 3 frequencies. This ES-induced vasoconstriction at 20 Hz and 40 Hz could be partly inhibited by H1 receptor antagonist chlorpheniramine (P < 0.05). After the exhaustion of histamine in mast cells by pretreatment with specific mast cell degranulator compound 48/80, chlorpheniramine could still inhibit the ES-induced flow rate reduction. In contrast, H2 receptor antagonist cimetidine could enhance the 40-Hz ES-induced flow rate reduction (P < 0.05). Moreover, ES-induced vasoconstriction at the 3 frequencies could all be enhanced by H3 receptor antagonist thioperamide (P < 0.05). Histamine could still be detected after the exhaustion of histamine in mast cells, and this histamine was co-released with NE. The concentration of HA was 0.52±0.71pmol/ml in 10Hz, 3.23±0.94 pmol/ml in 20 Hz, and 4.76±1.17 pmol/ml in 40 Hz. The release of HA increased with the increase of frequencies applied (P﹤0.01, 20 Hz vs 10 Hz; P﹤0.05, 40 Hz vs 20 Hz). These results indicate that stimulation on the auricular nerve may evoke histamine release from sympathetic nerves rather than from mast cells; that the release of histamine was frequency dependent to some extent; and that the functions of sympathetic histamine vary from pre-synaptic modulation to post-synaptic vasoconstriction or vasodilatation, via activation of different histamine receptors.In conclusion, 1) Histamine can be released from sympathetic nerves which modulate the peripheral vascular function in rabbit ear. 2) Sympathetic histamine can negatively modulate the vasoconstriction induced by symapathetic nerve stimulation via activation of presynaptic H3 receptors. 3) Sympathetic histamine can mediate vasoconstriction or vasodilation under different conditions via activation of H1 or H2 receptors, respectively.