Presynaptic H3 Receptors And α₂ Receptors Co-modulate Histamine Release From Sympathetic Nerves

We have found in our previous study that the enzyme for histamine (HA) synthesis (i.e., histidine decarboxylase) and the enzymes for norepinephrine (NE) synthesis (i.e., tyrosine hydroxylase and dopamine-β-hydroxylase) coexist in sympathetic neurons and that HA and NE also coexist in sympathetic neurons. Furthermore, depolarization with high concentration of K+ can trigger HA release from cardiac synaptosomes. These results indicate that HA is probably an unrecognized sympathetic neurotransmitter. However, in order to confirm that HA is a new sympathetic neurotransmitter, it should be clear that whether HA can release from sympathetic nerve terminals, whether the release pattern of HA is consistent with that of the classic sympathetic neurotransmitters and whether the presynaptic receptors can modulate the release of HA. Therefore, in order to clarify these problems, in this study isolated guinea pig vas deferens were perfused and stimulated with electrical field and the content of HA and NE released were detected with spectrofluorometer to provide further evidence to reveal the transmitter character of HA originated in sympathetic nerves.1 The establishment of modified fluorometric method for histamine determinationIn order to detect HA content in perfusate, we have modified and established a fluorometric method for histamine determination, respecting that some other methods are to some extent trivial, time consuming or relatively low sensitivity.O-phthalaldehyde (OPA) was used to derivatize HA released from tissues. Excitation and emission scan were performed on the reaction system. Na3PO4 was used to precipitate Ca2+ and Mg2+. The excitation maxima and emission maxima is 347 nm and 442 nm, respectively. Na3PO4 can diminish the inhibitory effect of Ca2+ and Mg2+ on the fluorescence intensity of the reaction system. The working curve for HA is F = 14.276C + 40.392 with the linear range of 1～100 ng/ml and with correlation coefficient of 0.9995. The detection limit is 0.5 ng/ml. The recovery is between 91.9%-115.8%. This method can be employed to detect HA in released from tissues without extraction of HA from the sample and is easy to operate.2 The effect of different frequencies of electrical field stimulation on HA release from sympathetic nervesEx vivo guinea pig vas deferens were stimulated with different frequencies of electrical field and the content of HA and NE in medium were detected with spectrofluorometer. When the frequency of electrical field stimulation increased, HA release also increased. However, the amplitude of the increase of HA release is higher than that of NE. The release of HA could be abolished by tetrodotoxin (TTX). The concentration of HA released in the perfused Krebs solution was sharply decreased from 1.16±0.11 to 0.14±0.08 pmol/ml·mg. In vitro denervation of vas deferens by 6-hydroxydopamine (6-OHDA) could also diminish HA release. The HA concentration decreased from 1.09±0.18 to 0.11±0.05 pmol/ml·mg. Pretreatment with the vesicular monoamine transporter 2 (VMAT2) inhibitor reserpine could diminish HA release from 1.11±0.13 to 0.27±0.12 pmol/ml·mg. It suggests that HA detected in these experiments was originated in sympathetic nerves. And the release of HA is more sensitive to high frequency of electrical field stimulation compared to the release of NE. The kinetics of the release of HA from nerve terminals may be different from that of NE.3 The modulation effect of presynapticα2 adrenoceptors and HA H3 receptors on HA releaseThe selectiveα2 adrenoceptor agonist brimonidine does-dependently decreased the release of both HA and NE, but the decreasing effect of NE was greater than that of HA. This inhibitory effect on HA release could be blocked by yohimbine, a selectiveα2 adrenoceptor antagonist. yohimbine used alone had facilitary effects on HA release. The results indicate that theα2 adrenoceptors on the presynaptic sympathetic nerve terminals have inhibitory effects on HA release. The HA H3 receptor agonist R-(α)-methylhistamine (αMeHA) also decreased HA release, which effect could be blocked by HA H3 receptor antagonist thioperamide, indicating that HA H3 receptors also have negative feedback effects on HA release. In addition, when HA H3 receptors were blocked by thioperamide, the inhibitory effect of brimonidine on HA release was strengthened. The inhibitory effect of HA release increased from 35±3% to 61±7% (n=5). while HA H3 receptors were activated byαMeHA, the inhibitory effect of brimonidine on HA release was diminished. The results suggest that the inhibitory effect ofα2 adrenoceptors on HA release may be influenced by the activating state of HA H3 receptors.In conclusion, 1) HA can release from sympathetic nerve terminals when sympathetic nerves are activated and the kinetics of the release of HA from nerve terminals may be different from that of NE. 2) Sympathetic neural HA release is modulated by both presynapticα2 adrenoceptors and HA H3 receptors. 3) HA H3 receptor may be a predominant receptor in negatively modulate HA release from sympathetic nerve terminals.