Biochem. Physiol. Vol. 92C, NO. 2, pp. 323-327,
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CATECHOLA~INE DEGRADATION IN THE H~~OLY~P~ OF THE CHINESE CRAB, ERIOCHEIR ~r~~~~~~ U. HOEGER and E. FLOREY Fakultiit fiir Biologie, UniverWt
Konstanz, 7750 Konstanz, Federal Republic of Germany. Telephone (0753 I) 88- 1 (Reeeiued 13 April 1988)
As determined by HPLC, the hemolymph of the Chinese crab, Eriocheir sinensis,contains low concentrations of DOPA (1.45 f 0.67 PM/ml) and noradrenaline (0.38 f 0.19 @/ml). Adrenaline and dopamine were not detectable (less than 0.1 PM/ml). 2. Clearance rates for the catecholamines DGPA, dopamine, noradrenaline and adrenaline were measured by HPLC after bolus injection (50 nM,/lOOg live weight) into the hemdymph. Half life of both DOPA and dopamine was about 7 min, that of noradrenaline and adrenaline between 27 and 40 min. 3. The results suggest a specificity of the cateeholamine removing system(s) towards the different catecholamines. The physiologica significance of the findings is discussed in the light of known actions of catecholamines on various crustacean organs.
It has long been peciaily dopamine, pharmacologically gans such as the receptor neurons
known that cat~holamines, esnoradrenaline and adrenaline, are active in various crustacean orheart, the hindgut and stretch (for an overview see Leake and
Walker, 1980). A catecholaminergic innervation of the hindgut has been described by Elekes et al. (1988), but nothing is known about similar innervation of other crustacean organs including the stretch receptor organs. The sensitivity of those organs found responsive to applied catecholamines would be sug gestive of a humoral control mechanism if it can be shown that one or the other of these catecholamines can occur in the hemolymph in physiologically significant concentrations. To our knowledge, the only report of catecholamines in crustacean hemolymph is that of Elofsson et al. (1982) who found hemolymph concentrations of 5 nM and 0.5 nM for dihydrox~henylalanine (DQPA) and noradrenaline, respectively, in the erayfish, Pacifastacus l~niasc~ias hemolymph. Dopamine was not detected and of other monoamines tested, detectable levels of octopamine (about IOnM) and serotonin (less than 0.5 nM) were found. With respect to the catecholamines, the values are lower than required to exert significant pha~acologica1 effects. Dopamine is the most active of all transmitter substances tested so far on isolated stretch receptor neurons of’ crayfish (McGeer et al., 1961; McLennan and Hagen, 1963). When tested on semi-isolated hearts of crustaceans, the positive chronotropic and inotropic effect was less than that of octopamine, noradrenaline or adrenaline (Florey and Rathmayer, 1980). On the crayfish hindgut, preliminary tests in our laboratory have shown that dopamine as well as noradrenaline and adrenaline exert similar stimulatory actions. The threshold concentrations of the amines can be as low as 10VgM in receptor neurons, and 10eBM in heart and gut. The steady state blood concentration of noradrenaline found in Pa~~~~tac~s 323
(Elofsson et al., 1982) is below that found effect% in isolated organs. Quantitative dete~inations had revealed octopamine, serotonin, noradrenaline and dopamine together with the precursor DOPA in different parts of the nervous system (Elofsson et al., 1982; Laxmyr, 1984; Elekes et a!., 1988). Cat~hoIamines and other biogenic amines might appear in the hemolymph as a result of neurosecretion as described for octopamine which is released from nerve terminals of the pericardial organs in the spiny lobster (Sullivan et al., 1977). In the light of these observations it was of interest to discover if there exist m~hanisms for the removal of catecholamines from the hemolymph after release into the circulation. Steady state concentrations in the presence of a removal mechanism would impfy more or less steady addition of catecholamines from nervous or non-nervous sources. Even a low hemolymph concentration might be representative of a steady state between release and removal. Upon sudden release, the concentration might rise momentarily to a much higher value provided inactivation or removal do not occur at rates high enough to prevent the build-up of concentrations to levels much greater than those of the steady state situation. Such a situation might allow dopamine to transiently reach detectable and physiolo~cally effective concentrations. Another origin for cat~holamines present in the hemolymph might arise from a situation similar to that in vertebrates where catecholamines arise by spillover of amines released from nerve terminals into circulation (Clare and Sever, 1982; Hjemdahl, 1987). The present investigation represents a first step in assessing the capacity of crustaceans to inactivate or remove catechol~ines from the blood stream. In this paper, we report clearance rates for the three catecholamines-adrenaline, noradrenaline and dopamine-as well as for the precursor DOPA in the Chinese crab, Eriocheir sinensis. Our rest&s suggest a specificity of the clearance mechanisms for different
U. HOEGER and E. FLOREY
catecholamines. The clearance rates are slow enough to permit a transient build-up of physiologically significant hemolymph concentrations. They are fast enough to explain the low steady state hemolymph concentrations actually found. MATERIALSAND
Chinese crabs were caught in the Elbe estuary near Hamburg using baited traps. They were communally kept in recirculated Lake Constance water and fed fish meat weekly. Male animals between 50 and 80 g wet wt were used for the experiments. The molting stage was not determined. However, most experiments were carried out between March and July, before the molting season which started in August in the laboratory population, For the catecholamine injections, the animals were held in a plastic lined laboratory clamp and kept partially submerged in a tray containing holding tank water with the last pair of walking legs exposed to the air. The animals were allowed to acclimate for 60-90 min prior to the catecholamine injections (see below). Catecholamine injections
Catecholamine solutions (1 mM in crayfish saline) were prepared immediately before use in crayfish saline, containing (in mM/l) NaCl(200), KC1 (5.4), MgCl, (2.6) and CaCl, (13.5) (Florey and Rathmayer, 1980). Solutions of DOPA were adjusted to pH 7.2-7.5 with 1 N NaOH. Injected volumes ranged between 25 and 35 ~1 to give a final dose of 50 nmol per 100 g live wt. This amount was chosen because lower doses of DOPA and dopamine were found to be cleared too rapidly from the hemolymph to allow precise measurements of the removal rates. Injections were done under the rear end of the center of the carapace. During the experiment, the animals were usually quiet except for occasional movements of the legs. Hemolymph collection
Hemolymph (7&90 ~1) was collected from the arthrodial membrane at the base of the 3 or 4 walking leg using a fine glass capillary. When drawing multiple samples, the capillary was always inserted at the same spot to avoid multiple penetration of the cuticle. The total amount of hemolymph removed from the animal during multiple sampling was 3-5% of the estimated total hemolymph volume. Catecholamine extraction The hemolymph was transferred into preweighed ice-cold 1.5 ml reaction vials containing 250 ~1 0.1% EDTA and 50 ~1 freshly prepared 0.1% sodium metabisulfite. The samples were processed immediately or kept at -20°C for not longer than 3 days before processing. The diluted hemolymph was deproteinized with 400 ~10.4 N PCA/O. 1% EDTA and centrifuged at 4°C for 2 min at 6000 g. TO the clear supernatant, 25mg Alumina prepared according to Anton and Sayre (1962) was added followed by the addition of 250 ~1 Tris-HCI buffer (2.5 M, pH 8.6) and 4 pM (50 ~1) of dihydroxybenzylamine in 0.1% EDTA as internal standard. The catecholamines were adsorbed by shaking for 10min at 4°C and the Alumina was washed with 1 ml of 0.1% EDTA/O. 1% sodium metabisulfite. After removal of the supematant, the catecholamines were desorbed with 50 ~10.25 N perchloric acid and 20 @Iwere injected into the liquid chromatograph (see below). For the determination of endogenous catecholamine levels in the hemolymph, [email protected]
~1 hemolymph was withdrawn, mixed with 50~1 of each sodium bisulfite (I%), EDTA (2%) and internal standard (4pM) and then deproteinized by addition of 100~1 2.4 M perchloric acid. After centrifugation, the supernatant was processed as described above.
HPLC determination of the catecholamines
The catecholamines were determined by ion pair high pressure liquid chromatography (HPLC) and electrochemical detection using a 250 x 4.6 mm reverse phase column (Nucleosil 5C,, , Macherey-Nagel Co., Dueren, FRG) and a mobile phase consisting of citric acid (100 mM), sodium acetate (50 mM), EDTA (25 mg/l) and sodium octyl sulfate (100 mg/l), pH 3.0. For the determination of DOPA, the concentrations of both citrate and acetate were halved. Noradrenaline was separated by anion exchange chromatography (Nucleosil SA, 4.6 x 200 mm) and a mobile phase consisting of 100 mM trisodium citrate and 36 mM sodium acetate, pH 5.2 (Elofsson et al., 1982). The oxidation voltage was set at 0.85 V. Dye injection
Kiton pure blue V (KPB V) was obtained from Chroma Gesellschaft, Stuttgart, and purified as described (Parekh et al., 1973). From a 0.25% stock solution in crayfish saline, 40-50~1 (2OOpg/lOOg live wt) were injected and samples taken as described above. The hemolymph was transferred to preweight reaction vials containing 700 ~1 1% EDTA and centrifuged at 6000g for 2 min. The dye concentration of the supematant was determined photometrically at 640 nm.
RESULTS Hemolymph samples obtained from 6 specimens were analyzed for adrenaline, noradrenaline, dopamine and DOPA. Adrenaline and dopamine were not detectable (less than 0.1 PM/ml hemolymph) but all samples contained low concentrations of DOPA (1.45 + 0.67 PM/ml; SD, N = 6) and noradrenaline (0.38 k 0.19 PM/ml; SD, N = 6). After the injections (catecholamines: 50 nM/lOO g live wt, KPB V: lOO~g/lOOg) both the dye and the catecholamines took about 5 min to reach maximal concentrations in the hemolymph. The disappearance of both dye and the catecholamines from the hemolymph followed an exponential decrease and linear regression analysis after In-transformation of the data yielded correlation coefficients with r > 0.98, indicating a first order type behavior. Half life (tli2) values were calculated from the expression ln2/k using the decay curve y = AekX, where x represents time (min) and y the measured catecholamine concentration in the hemolymph. A and k are constants, the latter being the decay constant. The tli2 values and decay constants are presented in Table 1. The fitted curves were extrapolated to the y-axis intercept (indicated by arrows in Fig. 1) which theoretically represents the maximally possible concentration of the injected compound in the hemolymph at time 0 min assuming complete equilibration. A mean value of 1.85 + 0.43 nM catecholamine per ml hemolymph was obtained (N = 8). From this value the dilution factor for the 50 nmol injected into a standard 100 g animal was calculated which was taken as an estimate of the hemolymph space. On a wet wt basis, a mean value of 27.7 + 6.4% of the wet wt was obtained (N = 8). In a few cases however, the calculated y-axis intercepts yielded much higher values around 3.5 nM/ml which suggests that the injected bolus did not always equilibrate completely with the total hemolymph space. However, no differences in the decay constants for the different catecholamines were
DOPA and NA in crab blood
Table 1. Decay constants and half lives for different catecholamines and kiton pure blue V after injection into the hemolymph of Eriocheir sinemis. Ambient temperature I 15°C Decay constant mean (range)
Half life (min) mean (range)
-0.108 (-0.102--0.113) -0.107 (-0.091--0.136) -0.026 (-0.023- -0.029) -0.021 (-0.017--0.023) -0.0058 (-0.003>-0.0088)
DOPA Dopamine Noradrenaline Adrenaline Kiton pure blue V
found in these experiments compared to the data listed in Table 1. The clearance rates (t,,?) were dependent on the kind of catecholamine injected: dopamine and DOPA were removed fastest with mean half life around 7min whereas noradrenaline had considerably longer half lives of more than 20 min. Adrenaline showed the lowest clearance rate with about 40 min half life. The chemical instability of the catecholamines at alkaline pH was also considered since the pH of the crab hemolymph is around 7.8 (unpublished results). Chemical decomposition was judged by incubating a 1 micromolar solution of catecholamines in crayfish saline in the dark at 15°C. Samples were taken every hour and analyzed by HPLC as described above. A decomposition rate of about 3% per hr was obtained under these conditions for all catecholamines. The chemical decomposition was therefore not considered significant compared to the clearance rate measured in the crabs. KPB V (mol. wt 567; Parekh et al., 1973) is a non-metabolizable dye that has been successfully used to determine clearance rates in vertebrates (Popa et al., 1974). We employed this substance to obtain estimates of hemolymph volume and to find the basal rate of loss of a hemolymph constituent that is, presumably, not removed by metabolic processes. The disappearance of KPB V from the hemolymph
6.4 6.7 26.9 38.5 134
(6.2-6.5) (5.1-7.6) (23.7-30.1) (30.5-40.3) (79-199)
N 2 3 3 3 5
also followed an exponential curve (data not shown) but was considerably slower than that of the catecholamines with a mean half life of more than 2 hr (see Table 1). Calculation of the hemolymph volume from the dye dilution obtained from the y-intercept of the extrapolated curves yielded values for the hemolymph space of 19.2% f 2.7% (SD; N = 5) of the live wt. Excretion of the dye into the surrounding water was observed but not determined quantitatively. DISCUSSION
From the extrapolation of the decay curves to the y-axis intercept (i.e. zero time) a maximum concentration of the catecholamines of 1.8 nM/ml hemo-
lymph was calculated which would correspond to a hemolymph space of 28% on a wet wt basis (see Results). This (theoretical) maximum was never reached suggesting that part of the injected catecholamines had already been removed before complete equilibration with the hemolymph. In contrast, the initial concentrations of KPB V were close to the extrapolated values due to the rather slow disappearance of the dye from the hemolymph. The mean value for the hemolymph space of 28% of the live wt calculated from the catecholamine dilution compares with a value of 26% obtained from the blue crab (Callinectes supidus) using 14Cthiocyanate as a marker (Gleeson and Zubkoff, 1977) and the mangrove crab Goniopsis cruentata using “C-inuline (Zanders, 1978). For the shore crab Curcinus maenas, a value of 33% (range 29-36%) was
Fig. 1. Clearance cf DOPA (A), dopamine (A), noradrenaline (0) and adrenaline (m) from the hemolvmoh of individual. Eriocheir sine&. after bolus inj&tibn (50 nmol/lOO g live wt). Broken parts of the curves represent interpolation to the y-axis intercept (indicated by arrows). Ambient temperature = 15°C.
determined (Siebers and Lucu, 1973) using the amaranth dye dilution method. Using 14C-inuline as marker in the latter study the extracellular space was found only between 17 and 19% of body weight (Siebers and Lucu, 1973) a value close to that calculated in this study on the basis of the KPB dilution. Although KPB has a much lower mol. wt than the polysaccharide inuline the lower values for the hemolymph space obtained in both cases suggests that the extracellular space available for a given molecule depends not only on its size but also on its chemical properties. This could be explained by differential distribution between different compartments of the extracellular space. Our observation, that in some cases the calculation of the extracellular space on the basis of the catecholamine dilution gave also smaller values around 15% of wet wt, might indicate that the extracellular space available for circulating catecholamines and other monoamines can vary depending on the physiological conditions. In crustaceans, the size of the hemolymph space is influenced by a variety of parameters including size, age and
U. HOEGERand E. FLOREY
molting stage (cf. Gleeson and Zubkoff, 1977) which were not considered in this study. Mechanisms of catechoiamine clearance
There are two possible routes of catecholamine removal from the hemolymph which are (1) the reabsorption and/or metabolism by appropriate tissues; and (2) the removal by excretory processes via the nephridial organs. The comparatively rapid removal of the catecholamines with half lives between 10 and 40min contrasts strongly with the slow disappearance of KPB V and the persistence of the metabolically inactive compound thiocyanate found earlier (Gleeson and Zubkoff, 1976) suggesting that the catecholamine clearance is due to removal processes independent of excretion. Little information exist on the persistence of other neuroactive compounds in crustacean hemolymph. In the lobster, the neuropeptide proctolin is cleared from the hemolymph with a half life between 610min which is in the same range as found for DOPA and dopamine in this study (Schwarz et al., 1984). Circulating serotonin injected into the hemolymph of Carcinus maenas showed a half life of less than 25 min (personal communication, cited in Mattson and Spaziani, 1986). In comparison, the half lives for glucose found after injection into the hemolymph of the crab, PachJgrapsus crassipes were considerably longer and showed a temperature dependent time course of disappearance with half lives between 1 and 3 hr (Johnson and Schatzlein, 1981). The time course of catecholamine degradation in the hemolymph might be influenced by the experimental conditions. (1) The stress due to experimental handling might lead to an increase in the animal’s basal heart rate as reported for the shore crab (Wilkens et al., 1985). (2) The action of injected catecholamines on cardiac performance has further effects both on heart rate and heart contraction, and hence on circulation rate which in turn might influence the catecholamine clearance. (3) When tested on semiisolated heart preparations of Astacus leptodactylus and Eriphia spinifrons (Florey and Rathmayer, 1978) the cardio-accelerating effect of dopamine was much stronger (6&100 times in Astacus and 6-20 times in Eriphia) than that of both noradrenaline and adrenaline. The different clearance patterns could therefore be related to the different effect of the catecholamines on the heart performance. This is, however, not likely: in preliminary experiments it was found that heart rates of Chinese crabs injected with dopamine or noradrenaline increased with a subsequent decrease similar to the decay curves found in this study. On the other hand, injected DOPA did not produce a significant effect on the heart rate although its clearance characteristic was found identical to that of dopamine (see Table 1). Injected noradrenaline, on the other hand, did increase the heart rate although its clearance was much lower than that of both DOPA and dopamine. These findings suggest that the observed clearance patterns are related to the specificity of the removing system towards different catecholamines. The processes related to catecholamine reuptake and/or metabolism might also be
subject to the physiological state of the animal and therefore represent a possible control system for the regulation of the persistence of catecholamines in the hemolymph. There is only little information about the ultimate fate of these compounds and the exact site(s) of catecholamine uptake and possible pathways of metabolism. In crustaceans and other invertebrates, monoamines are inactivated by N-acetylation and/or sulfatation as demonstrated for nervous and other tissues (Hayashi et al., 1977; Kennedy, 1978), but both monoamine uptake and metabolism in craytish might also occur in tissues such as the digestive gland, an organ which is in close contact with the circulating hemolymph. The hemolymph itself does not seem to play a role, since at least octopamine has not been found to be metabolized by the hemolymph of Panulirus interruptus). Catecholamine turnover
Under steady state conditions, the rates of catecholamine release into and removal from the hemolymph are equal and represent the turnover rate. Our experiments do not allow the direct determination of the turnover rate since this requires steady state concentration of the respective metabolite in the hemolymph. The amount of hemolymph (7&90~1) drawn in repetitive samples from an animal was not sufficient for the determination of basal catecholamine concentrations. For a tentative calculation, we applied the respective decay constants (see Table 1) to the hemolymph levels of DOPA (1.45 PM/ml) and noradrenaline (0.4 PM/ml) found in the Chinese crab. Although the decay constants were measured with experimentally elevated catecholamine concentrations, the catecholamine clearance showed a logarithmic relationship over a wide concentration range which indicates that the removing system was not saturated and thus allows extrapolation to the lower values. In the case of saturation, a linear clearance characteristics would have been obtained. Based on these values, a DOPA clearance rate of 0.15 PM/ml per min is obtained and this rate would have to be balanced by an equivalent release of DOPA into the hemolymph to maintain steady state concentration. In the same way, a value of about 0.01 PM/ml per min is calculated for noradrenaline. For a standard 100 g crab with a hemolymph space of 28 ml, about 4 pM DOPA and 0.3 pM noradrenaline would therefore be released (and removed) per minute into the hemolymph at 15°C. If there were a momentary neurosecretory dopamine release into the hemolymph to concentrations that have been shown to exert significant effects on the heart in Astacus (i.e. 10-8-10-9 M; Florey and Rathmayer, 1978) the half life of 7 min would reduce the dopamine level below threshold (lo-” M) in about 40min on the basis of our experimental data. The large apparent removal capacity of the Chinese crab for catecholamines at concentrations one thousand-fold above normal is surprising in the light of their normally low concentration in the hemolymph. The noradrenaline levels found in the Chinese crab compare with those found in the plasma of fish (e.g. Boutilier et al., 1988) and an amphibian (Tufts et al., 1987) under resting conditions. One
DOPA and NA in crab blood explanation could be that in vivo elevated concentrations of catecholamines occur only locally and are removed again soon after reaching the target organ. This situation has been suggested for the action of
octopamine and serotonin on the modulation of posture in the lobster (see Beltz and Kravitz, 1987). The “strategic” position of a removing system would therefore confine the action of an active compound on a limited part of the circulating system and in the case of dopamine could also explain its low concentration in the hemolymph which was below the detection limit (
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