심근의 Catecholamine Uptake에 관한 연구

Abstract

[한글]

[영문]

Loewi's classical experiments in 1921 which demonstrated the presence of sympathetic neurotransmitter in the heart, have laid the groundwork for understanding of the chemical transmission of sympathetic nerve impulses. It is now well established that the transmitter of sympathetic nerve is identical with norepinephrine (Barger and Dale, 1911; Bacq, 1934; Euler, 1948).

During the following decades a considerable amount of interest was directed to the synthesis, storage, release and metabolism of catecholamines both in the sympathetic nerve and in the adrenal medulla. A considerable proportion of the catecholamines in the adrenergic nerve is kept in intracellular particles and these

storage granules have a very high content of adenine nucleotides (Falk et al.,1956; Schumann, 1958b; Potter and Axelrod, 1963b). The remarkable constancy of the molar catecholamine/adenine nucleotide relationship under various conditions has led to the proposition that the catecholamine stored in the granules actually form a complex with adenine nucleotides and some third component, probably intragranular protein (Carlsson and Hillarp, 1956a; Blaschko et al., 1957). Within these granules which contain the enzyme dopamine beta-oxidase, the last step in the biosynthesis of norepinephrine (the beta-hydroxylation of dopamine), has been shown to occur (Potter and Axelrod, 1963b). On the other hand, the decarboxylation of dopa takes place in the cell sap (Masuoka et al., 1958). While catecholamines are synthesized and stored locally both in adrenergic nerves and in the adrenal medulla, recent investigations have clearly shown that they are also released from the postganglionic sympathetic nerve endings and a part of these released norepinephrine as well as injected norepinephrine is to a variable extent taken up

into tissue stores (Raab and Gigee, 1955; Muscholl, 1960). Furthermore, the existence of a highly efficient mechanism for uptake and retention of circulating catecholamines in the tissues has been demonstrated by following the fate of intravenously injected radioactively labeled epinephrine (Axclrod et al., 1959) and norepinephrine (Whitby et al., 1961).

Nevertheless of these evidences, little is known about the uptakeability of several catecholamines which are structurally analogous to norepinephrine or epinephrine. In this respect, it was of interest to examine the nature and the relative potency of uptake among these catecholamines when the uptake was actually

involved in the heart and to explore the role of enzymes (MAO and COMT) in the process of catecholamine uptake. In addition, it was also designed to determine the catecholamine uptake by chronically denervated heart.

The experiments were conducted on the isolated atria. The catecholamine content in atria was determined by the spectrophotofluorometric procedure described by Shore and Olin (1958) and the monoamine oxidase activity was determined colorimetrically according to the procedure of Green and Haughton (1961), using mitochondria prepared from cardiac homogenates in 0.25 M sucrose.

Results

1. The intraperitoneal injection of reserpine (3mg/kg) into rabbits 24hr prior to study, abolished the response of the atria from these animals to tyramine (5×10**-5 M) a concentration which produced a marked increase in the rate and contractile amplitude of atria from normal rabbits. The atria from rabbits

pretreated with reserpine were exposed for 10 min to various concentrations of norepinephrine. Forty-five min after removal of the bathing fluid containing norepinephrine and following repeated exposure to fresh bathing fluid, the response

of atria to 5×10**-5 M of tyramine was partly restored. Exposure to concentrations of 3×10**-6 M or greater of norepinephrine increased slightly, but significantly the catecholamine content. Also, the magnitude of the restored response to tyramine and the increase of catecholamine content of the atria appeared to depend on the concentration of norepinephrine to which the atria were exposed.

2. Atria from rabbits pretreated with reserpine were exposed to various concentrations of epinephrine, isoproterenol or alpha-methylnorepinephrine (cobefrine) for 10 min and their respinse to tyramine were then determinded.

Epinephrine partially restored the response to tyramine but the magnitude of the response was much smallar than that following exposure to norepinephrine. After treatment with isoproterenol it was not possible to demonstrate any restoration of the response to tyramine. Cobefrine, however, produced more striking restoration of the response to tyramine than did norepinephrine.

3. Before exposure of the atria from reserpinized rabbits to norepinephrine they were incubated in SKF-385 (10**-5 M) for 30 min. The response of these atria to tyramone was compared with that observed with atria which were exposed to the same concentration of norepinephrine but not SKF-385. Tyramone produced a more marked increase in contractile amplitutue of atria which were treated with SKF-385 before exposure to norepinephrine than those exposed to norepinephrine only. JB-516

(10**-4 M) and iproniazid (10**-3 M) significantly enhanced the respone to tyramine produced by prior exposure to norepinephrine. SKF-385, JB-516, and iproniazid significantly increased the amount of norepinephrine accumulated by the atria.

Inhibitory activities of SKF-385, JB-516 and iproniazid in the concentrations employed in these experiments, on the MAO of mitochondria prepared from homogenates of rabbits hearts were determined. The magnitude of cardiac MAO inhibition appears

to be related to the magnitude of the enhancement of accumulation of norepinephrine produced by these MAO inhibitors.

4. Atria from reserpine pretreated rabbits were exposed to SKF-385, JB-516 or iproniazid for 30 min and then to cobefrine for 10 min. Forty-five min after removal of cobefrine from the bath and following repeated changes of the bathing fluid, the response of atria to tyramine was compared to that of atria treated identically except that a MAO inhibitor was not added prior to the cobefrine. Nether SKF-385, JB-516 nor iproniazid had an effect on the restoration by cobefrine of the response to tyramine.

5. Before exposure of the atria from reserpinzed rabbits to norepinephrine they were incubated in pyrogallol (3×10**-5 M) for 30min. The response of atria to tyramine was compared to that of atria treated identically except that and the content of atrial catecholamine were practically same as those observed in the atria prior to administration of pyrogallol.

6. Pretreatment of the atria with bretylium (5×10**-4 M) for 30 min markedly inhibited their uptake of catecholamine following exposure to norepinephrine.

7. Bilateral sympathectomy in cats 15 days prior to study, markedly depleted the myocardial catecholamine content and abolished the response of the atria from these animals to tyramine. The exposure of norepinphrine to the atria failed to restore both the cardiostimulant action of tyramine and to increase the catecholamine content of these atria.

From the above results, it is obvious that the catecholamines, when added in vitro to isolated atria pretreated with reserpine, cause the restoration of cardiostimulant response to tyramine and the increase of cardiac catecholamine contents. Furthermore it may be concluded that MAO and the intact sympathetic nerve play an important role in the accumulation of norepinephrine by the heart, thereby regulating the level of cardiac catecholamines.