pericardial sinus caused a slight decrease in heart rate concurrent with a large increase in cardiac stroke vol- ume. It decreased haemolymph flow anteriorly ...
J Comp Physiol B (1994) 164:103-111
Journal of Comparative ~...,o,"~"~ Physiology B -'"
and EnvironPhysiology
9 Springer-Verlag 1994
Peptidergic modulation of cardiovascular dynamics in the Dungeness crab, Cancer magister I.J. McGaw 1,2, C.N. Airriess t,2, B.R. McMahon 1,2 1Department of Biological Science,Universityof Calgary, Calgary,Alberta, T2N 1N4, Canada 2 BamfieldMarine Station, Bamfield,British Columbia, V0R 1B0, Canada Accepted: 8 February 1994
Abstract. Decapod crustacean pericardial organs contain extensive neurohormonal reserves which can be released directly into the haemolymph to act as physiological modulators. The present paper concerns the in vivo effects of two pericardial peptides, proctolin and crustacean cardioactive peptide, on cardiovascular dynamics in the crab Cancer magister. Infusion of proctolin into the pericardial sinus caused a slight decrease in heart rate concurrent with a large increase in cardiac stroke volume. It decreased haemolymph flow anteriorly through the paired anterolateral arteries and increased flow posteriorly and ventrally through the posterior aorta and sternal artery, respectively. The threshold for responses occurred at circulating concentrations of 10 -9 mol.1 -~, and haemolymph flows remained elevated for up to 30 min after peptide infusion. The effects of crustacean cardioactive peptide were less dramatic. Heart rate was not affected but a significant increase in stroke volume was observed. Crustacean cardioactive peptide increased haemolymph flow through the anterolateral arteries and increased scaphognathite rate. The threshold for crustacean cardioactive peptide activity was higher than for proctolin (10 -7 tool.1 1 and 10 .6 tool.1-1) but the responses to crustacean cardioactive peptide were of longer duration. The effects of proctolin on regional haemolymph distribution in Cancer magister closely resemble the cardiovascular responses of this species when exposed to hypoxic conditions. These peptides may be implicated as cardiovascular regulators during environmental perturbations. Key words: Proctolin - Crustacean cardioactive peptide Cardiovascular - Circulation - Crab, Cancer magister
Abbreviations: CCAP, crustacean cardioactive peptide; CNS, central nervous system Correspondence to: I.J. McGaw
Introduction Until recently the open circulatory system of even the more advanced crustaceans was regarded as a simple system allowing little or no control over cardiac output and regional haemolymph distribution (Maynard 1960). More contemporary authors (McMahon and Burnett 1990) have suggested that the circulatory system of higher-order crustaceans is more complex and more efficient than previously described and is capable of tissue perfusion levels equivalent to those of some vertebrate systems. The decapod crustacean heart consists of a single muscular ventricle suspended within a second chamber, the pericardial sinus, by 11 suspensory ligaments. Five arterial systems (seven arteries) leave the heart to supply distinct body regions (Pearson 1908). These branch into fine capillary-like vessels which empty into tissue lacunae. Haemolymph then drains into the infra-branchial sinus before passing through the gills and back to the lateral pericardial sinus. Each main artery has a muscular, innervated valve at its origin (Alexandrowicz 1932) which may respond to neurohormonal agents in vitro (Kuramoto and Ebara 1984, 1989). The pericardial organs, situated in the pericardial sinus lateral to the heart and near the openings of the branchiocardiac veins (Alexandrowicz 1953), are the largest neurohaemal release sites in crustaceans (Stangier et al. 1987). Neurohormones released from these organs pass directly to the heart and cardiac ganglion via incurrent haemolymph flow and travel to more remote target organs via the arterial system (Alexandrowicz and Carlisle 1953). Substances extracted from the pericardial organs and found to have both chronotropic and inotropic effects on the heart (Cooke and Sullivan 1982) have subsequently been identified as the biogenic amines serotonin, dopamine and octopamine (Florey and Rathmayer 1978; Sullivan 1978). These amines have both excitatory and inhibitory effects on the heart (Florey and Rathmayer 1978; Airriess and McMahon 1992) and cardioarterial valves (Kuramoto and Ebara 1984) of decapod
104
I.J. MeGaw et al. : Peptidergic modulation of cardiovascular dynamics in a crab
crustaceans. A n additional role in regional b l o o d partitioning in vivo for C. magister has been p r o p o s e d (Airriess a n d M c M a h o n 1992). Several peptides have also been identified in pericardial o r g a n extracts. The pentapeptide proctolin was originally isolated and sequenced f r o m the h i n d g u t o f the c o c k r o a c h Periplaneta americanus (Starratt and B r o w n 1975). It was discovered by Sullivan (1979) in the pericardial organs o f the crab Cardisorna carnifex and has since been s h o w n to occur in a n u m b e r o f other crustaceans (Schwarz et al. 1984; Stangier et al. 1986) as well as other invertebrate phyla ( O r c h a r d et al. 1989). The cardiovascular and neural effects o f proctolin are varied and it m a y show species specificity even within the decap o d crustacea (Pasztor and Macmillan 1990; Bishop et al. 1991). The p r i m a r y effect o f proctolin on the heart is inotropic, increasing contractility o f isolated hearts o f Portunus puber and Homarus gammarus ( C o o k e and Sullivan 1982) a n d Carcinus maenas (Wilkens a n d M c M a h o n 1992). In intact C. maenas proctolin also increases ventilation rate (Wilkens et al. 1985) by its direct action on ventilatory muscle (Mercier and Wilkens 1985). Proctolin also has c h r o n o t r o p i c effects but these are m o r e variable. In intact C. maenas, injection o f proctolin causes slight t a c h y c a r d i a at concentrations below 10 . 7 m o l . 1 - 1 but higher doses cause mild b r a d y c a r d i a (Wilkens et al. 1985; Stangier et al. 1986; Saver and Wilkens 1992). In the lobster Homarus americanus proctolin has little effect on heart rate but plays a role in selective h a e m o l y m p h distribution. H a e m o l y m p h flow anteriorly t h r o u g h the anterolateral arteries and anterior a o r t a decreases while flow ventrally t h r o u g h the sternal artery increases ( M c M a h o n and Reiber 1991; M c M a h o n 1992), p r e s u m a b l y as a result o f peptide action on the cardioarterial valves. C C A P was first isolated a n d sequenced f r o m the pericardial organs o f the shore crab Carcinus maenas (Stangier et al. 1987) and is k n o w n to have c h r o n o t r o p i c effects on the isolated hearts o f d e c a p o d s (Sullivan and C o o k e 1982). It is a unique n o n a p e p t i d e with a disulphide bridge. Recently its presence has also been detected in the C N S o f the insects Locust migratoria (Stangier et al. 1989; Dircksen et al. 1991) a n d Tenebrio molitor (Breidbach a n d Dircksen 1991). Crustacean cardioactive peptide has b o t h c h r o n o t r o p i c a n d inotropic effects o n the heart of C. maenas (Stangier 1991 ; Saver and Wilkens 1992; Wilkens a n d M c M a h o n 1992), and similar findings have been reported for the crayfish Orconectes limosus (Stangier 1991). In intact Cancer pagurus, however, C C A P h a d no effect on the heart rate but did elicit long-term increases in scaphognathite beat freq u e n c y (Stangier 1991). In Carcinus the pericardial organs c o n t a i n a six- to sevenfold higher c o n c e n t r a t i o n o f C C A P c o m p a r e d with proctolin, and release o f < 1% o f this a m o u n t into the h a e m o l y m p h w o u l d be sufficient to effect changes in heart activity (Stangier et al. 1987). There have been very few reports o f peptidergic m o d ulation in intact Crustacea, especially with respect to cardiac o u t p u t a n d selective h a e m o l y m p h distribution. The present investigation, therefore, s o u g h t to determine the effects o f these two pericardial peptides on regulation
and control o f cardiac o u t p u t and regional h a e m o l y m p h distribution in vivo in the Dungeness crab, Cancer magister.
Materials and methods Adult male Cancer magister 500-800 g body weight were trapped in the Barkley Sound region of Vancouver Island, British Columbia, and held at Bamfield Marine Station in filtered running seawater at 12___1 ~ in a salinity of 33+_1 mg-ml 1 for at least 1 week prior to experimentation. Crabs were normally fed fish hi-weekly but were isolated from food supplies for 2 days prior to experimentation. Only crabs in intermoult were selected for experimental treatment. A 545C-4 directional pulsed-Doppler flowmeter (Bioengineering, University of Iowa) was used to measure haemolymph velocity in each of the major arteries. This technique of minimally invasive flow measurement is described in detail in Airriess and McMahon (1994). Briefly, piezoelectric crystal probes (Iowa Doppler Products, Iowa City, Iowa, USA and Crystal Biotech, Hopkinton, Mass., USA) were implanted in grooves abraded to the dermis of the carapace directly above the anterior aorta, left anterolateral artery, right hepatic artery and posterior aorta. Probes were focused to obtain maximum signal amplitude and then fixed in place using cyanoacrylate glue and dental wax. Haemolymph velocity in the sternal artery was measured via an internal catheter-mounted probe which was introduced through the first abdominal segment and guided along the median plate of the endophragmal system to the first descending portion of the artery; haemolymph loss during this procedure was negligible. Output from the flowmeter was recorded on a 6-channel oscillograph (Gould, Valley View, Ohio, USA). Heart rate was determined by counting the peaks corresponding to cardiac systole on the arterial flow traces. This method, evaluated by Airriess and McMahon (1994), gives identical results to the more familiar impedance conversion technique as long as there is haemolymph flow through at least one arterial system. Cardiac output was calculated by summation of the mean flow through each artery (values for paired arteries were doubled as simultaneous recording showed there to be no detectable difference in flow rates between left and right vessels), and this value was divided by mean heart rate to obtain cardiac stroke volume. Scaphognathite beat frequency was recorded using a hydrostatic pressure transducer (Statham/Gould P23Db, Hato Rey, Puerto Rico) connected via a saline-filled polyethylene catheter to the right branchial chamber. During experiments crabs were held in a 28 • 20 • 10 em Perspex box with a constant flow of aerated sea water. Following instrumentation, animals were allowed at least 24 h to settle before experimentation. Experiments were carried out at a temperature of 12_+ 1 ~ in constant darkness. Proctolin and CCAP (Peninsula Laboratories) were dissolved in Cancer saline (Morris and McMahon 1989) and diluted to achieve final calculated circulating concentrations of 10 -610-12 tool. 1-1. Test solutions were infused directly into the lateral pericardial sinus via a chronically implanted polyethylene catheter (PE20). A syringe pump (Sage Instruments) was used to infuse 350 gl of the test solution followed by 150 gl saline for catheter washout over a 3-rain period. This interval was long enough to ensure that the hormone did not reach the pericardial sinus as a concentrated bolus of injectate but rather allowed slow and equal distribution of the peptide solution (Airriess and McMahon 1992). Control infusions were carried out using Cancer saline in place of the test hormone. Each experimental animal received the entire concentration range of one of the hormones only. Haemolymph flow rates, heart rate and scaphognathite beat frequency were recorded at 10-rain intervals during a 30-min control period, during and immediately after infusion of either saline or peptide solution, and at regular intervals after infusion up to a total time of 120 rain for each concentration.
I.J. McGaw et al. : Peptidergic modulation of cardiovascular dynamics in a crab
and Uglow 1977). Within individual crabs, however, haemolymph flow rates and heart and scaphognathite beat frequencies remained relatively stable during the 30-min pre-treatment interval. These values are in accordance with those previously reported for C. magister resting in well-aerated sea water (Airriess and McMahon 1994). There was no significant change in any parameter associated with infusion of saline (P > 0.05; Figs. 1-4 a and c). Proctolin infusion at 10-8mol.1-1 caused a slight decrease in heart rate (F=2.18, P 0.05) for up to 20 min after infusion of proctolin at doses of J0-gmol.1-1 and above (Fig. 2b). The most dramatic change associated with proctolin
In order to determine whether peptide infusion caused a change in any of the recorded variables, data were subjected to a univariate analysis of variance with repeated measures design. Missing values were statistically estimated (Zar 1984). ANOVAs showing a significant treatment effect (P < 0.05) were further analyzed using Tukey's HSD multiple comparison test (Zar 1984) with ~ = 0.05.
Results
Arterial haemolymph flows as well as heart and scap'hognathite rates (mean+SEM), were determined for 10' animals tested with proctolin and l l animals tested with CCAP (Figs. 1-4). Each figure shows the mean responses to a control infusion of C. magister saline and a subsequent infusion of a test peptide at sufficient concentration to demonstrate clear modulatory ability. In general, the effects of proctolin were more dramatic than those of CCAP and the time-course of proctolin activity was shorter. Considerable inter-animal variability was observed in all measured cardiovascular parameters, as is common among conspecific decapod crustaceans sub,jected to identical laboratory conditions (Cumberlidge 1oo
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Fig. 1. a, b Mean ( + SEM) heart rate before, during and after infusion of 350 gl saline and 10-s mol-1-1 proctolin, c, d Mean (-t-SEM) haemolymph flow through the left anterolateral artery after infusion of 350 gl saline and 10 -8 m o l d - ~ proctolin. Infusions took place over a 3-rain period starting at 0 rain
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106
I.J. M c G a w et al. : Peptidergic modulation of cardiovascular dynamics in a crab 100
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Fig. 4a-d. Mean ( • haemolymph flow through (a, b) the left anterolateral artery and (e, d) the sternal artery after infusion of 350 gl saline and 10 7 m o l . l - 1 CCAP
infusion occurred in the sternal artery, which showed an increase in haemolymph flow velocity of over 300% (F=9.51, P0.05; Fig. 3 b). Crustacean cardioactive peptide did, however, affect the pattern of arterial haemolymph flow. Infusion of this hormone caused a marked increase in haemolymph flow through the anterolateral arteries (F=/.98, P 0.05; Fig. 4d). The haemolymph flow rates in all of the other arterial systems were highly variable and showed no characteristic response to CCAP infusion. There was a significant increase in scaphognathite rate associated with CCAP infusion (F= 3.22, P < 0.01). This reached a maximum of 100 +_10 beats, min-1 with-
I.J. M c G a w et al. : Peptidergic modulation of cardiovascular dynamics in a crab 400
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of each hormone. Proctolin significantly altered haemolymph flow through the sternal artery at concentrations of 10 -9 mol-1 -~ and above (Fig. 5a). Peak response occurred at 10- s tool. 1-1, at which concentration a 310% increase in flow through the sternal artery occurred. At higher concentrations the response magnitude declined. The dose-response curve for CCAP activity was bimodal and much less dramatic than that of proctolin (Fig. 5 b). There was a small increase in scaphognathite beat frequency associated with CCAP dosages of 10 - i t and 10-so tool.l-l, then no response at successively higher concentrations below I0-7mo1.1-1. At 10 - 7 and 10- 6 mol. 1- t the increase in scaphognathite frequency was dose dependent.
Calculated parameters
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Concentration (mol'1-1) Fig. 5a, b. Dose-response curves for proctolin (sternal artery) and CCAP (ventilation rate). Graphs represent means (-I-SE) for 10 animals tested with proctolin and 11 animals tested with CCAP, calculated as a percentage change (from pre-injectate values) during the first 10 min after initial hormone infusion
in 10 min of treatment and returned to pre-treatment levels of approximately 77 beats-min -1 after 30 min (Fig. 3 d). Dose-response curves for proctolin (sternal artery) and CCAP (scaphognathite frequency)/ire presented in Fig. 5. These variables were chosen because they most clearly represent the changes associated with infusion
Cardiac output was calculated by summation of the mean flows of all vessels leaving the heart. This allowed the arterial flow rates to be expressed as a percentage of cardiac output. In pre-treatment animals and those given a control injection of saline, approximately 40% of the haemolymph pumped by the heart was delivered via the sternal artery, while another 40% went to the paired anterolateral arteries (Fig. 6). Of the remaining 20%, approximately 9% of cardiac output was channeled through the paired hepatic arteries, 8% flowed through the posterior aorta, and 3% was delivered through the anterior aorta. The percentage of cardiac output delivered via each of these vessels remained relatively constant during and after the saline control injection. In contrast, administration of proctolin at 10 .8 mol.1-1 caused the pattern of cardiac output distribution to change drastically (Fig. 7). The percentage of cardiac output delivered to the sternal artery almost doubled (to 80%) after 3 rain and remained elevated for up to 25 rain after hormone treatment. This was coupled with a 75% decrease in allocation to the anterolateral arteries. Pre-infusion values were also regained after 25 min. The percentage of cardiac output delivered through the hepatic arteries showed a similar trend to that of the anterolaterals, decreasing in response to pro-
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Fig. 6. Change in percentage of cardiac output delivered to each artery after infusion of 350 gl saline, calculated as a percentage of total mean cardiac output
108
I.J. McGaw et al. : Peptidergic modulation of cardiovascular dynamics in a crab
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ctolin treatment. The percentage distribution through the anterior and posterior aortae was rather more variable and showed no detectable change in response to proctolin infusion. Infusion of C C A P also caused changes in the partitioning of cardiac output between the m a j o r arteries. The changes resulting from C C A P infusion at 10-7 mol. 1-1 differed sharply f r o m those associated with proctolin infusion and were of longer duration (Fig. 8). There was a slight decrease in the percentage of h a e m o l y m p h delivered through the anterior aorta. The percentage of cardiac output delivered through the paired anterolateral arteries increased dramatically after 3 min and reached a m a x i m u m of 70% within 10 rain of C C A P infusion. This change was concurrent with a 20% decrease in the per-
centage of cardiac output delivered via the sternal artery. The distribution did not regain its pre-injection pattern until 90 min after C C A P treatment. The percentage of cardiac output delivered to the hepatic arteries was more variable, showing no obvious trend, while distribution to the posterior aorta remained relatively stable. Cardiac stroke volume was calculated by dividing mean cardiac output by mean heart rate. Saline was found to have no effect on the stroke volume of the heart; however, proctolin (10 -8 mol.1-1) and C C A P (10- 7 mol. 1-1) increased stroke volume for up to 30 min from approximately 0.072 ml per beat to 0.155 ml per beat and 0.127 ml per beat, respectively (Fig. 9).
I.J. M c G a w et al. : Peptidergic m o d u l a t i o n o f cardiovascular dynamics in a crab 0.16 I.,..,
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Discussion
The primary reported effect of proctolin on isolated or semi-isolated hearts is its inotropic excitation [reviews: Cooke and Sullivan (1982); Wilkens and McMahon (1992)]. The present study confirms these observations for the in vivo heart of C. rnagister and also shows that the increase in heart tonus is associated with increased stroke volume. This increase is the only contributor to the observed increase in cardiac output since no increase in heart rate occurred. In the present study proctolin also had diverse effects on haemolymph flow through several arterial systems. Flow through the anterolateral arteries decreased despite the increase in cardiac output. The percentage of cardiac output delivered via the sternal artery increased (Fig. 7), but the magnitude of the increase exceeded that suggested by the increase in stroke volume. This implies that the haemolymph that would have passed into the anterolateral arteries was diverted to the sternal arterial system. Flows through all of the other systems changed relatively little; thus, the overall effects of proctolin on the heart of C. magister in vivo were to increase and redistribute cardiac output in order to elevate haemolymph flow through the sternal artery. This artery supplies the limbs, mouthparts and CNS of decapods via its subsidiaries, the ventral thoracic and ventral abdominal arteries (McLaughlin 1983). The anterolateral and hepatic arteries supply the carapace, testes, foregut and hepatopancreas. Therefore, the principle effect of shunting of haemolymph to the sternal artery is to increase perfusion of the CNS and the locomotor structures. This is functionally similar to the cardiovascular adjustments which occur during activity in mammals, where increased cardiac output is diverted away from visceral structures to the locomotor muscles. The threshold for cardiovascular responses to proctolin was approximately 1-10-9 mol. 1-1 with maximal effects occurring between 10 -s and 10-7mo1.1-1. This
109
is essentially similar to other arthropod neuromuscular preparations (Benson etal. 1981; Watson etal. 1983; Schwarz et al. 1984; Groome et al. 1990). In the present study proctolin did not act in a simple dose dependent manner. At the higher concentrations tested the effects of increasing dosages were of smaller magnitude (Fig. 5a) as has been reported for other arthropod preparations (Benson et al. 1981; Wilkens et al. 1985). Effects on blood flow were seen within 1 min of the start of infusion and often persisted for 10-30 min, a time-course similar to that reported by Watson et al. (1983) and Groome et al. (1990) for isolated Limulus hearts. However, shorter time-courses have also been reported for proctolin activity on Carcinus rnaenas hearts, both in vivo and in situ (Wilkens etat. 1985; Saver and Wilkens 1992). The inotropic effects of proctolin are thought to be mediated by its direct action on the cardiac muscle (Benson et al. 1981; Wilkens and McMahon 1992) rather than by affecting the cardiac ganglion: the increased stroke volume observed here probably resulted from this direct action of proctolin on the myocardium. Variable partitioning of cardiac output between the arterial systems is thought to be brought about by the contraction or relaxation of the muscular, innervated cardioarterial valves (McMahon and Burnett 1990). Procto!in depolarizes and causes contracture of the posterior valve muscle of isolated lobster (Panulirus japonicus) hearts, reducing systolic pressure in the posterior aorta and preventing backflow of haemolymph into the heart during abdominal contractions (Kuramoto and Ebara 1984). In the present study, proctolin did not decrease haemolymph flow through the posterior but caused a dramatic increase in haemolymph flow directed ventrally via the sternal artery. A similar response to proctolin, suggesting relaxation of the valve at the origin of this system, has been observed in the lobster Homarus americanus (McMahon and Reiber 1991). Anatomical differences in both the arrangement and functional relationship between the valves of the sternal artery and posterior aorta may account for differences in haemolymph redistribution among these decapods. Species specificity of proctolin activity may also be responsible for the discrepancies between isolated P. japonicus hearts and intact C. magister hearts reported here. This underscores the importance of studying whole animal preparations in addition to isolated or semi-isolated hearts: it is possible that studies involving isolated organ preparations may obscure important secondary messenger systems (Groome and Watson 1989). Proctolin also has direct excitatory effects on ventilatory muscle (Mercier and Wilkens 1985), bringing about an increase in scaphognathite beat frequency (Wilkens et al. 1983, 1985). In the present study, a slight increase in scaphognathite rate was observed at the two highest concentrations tested (10 .7 and 10-6mo1.1-1), but there was so much variability even during pre-injection periods th~/t definite conclusions cannot be drawn. Although the actions of CCAP on the cardiovascular system of decapods are less well understood than those of proctolin, this peptide is known to exert a strong
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1.J. McGaw et al. : Peptidergic modulation of cardiovascular dynamics in a crab
chronotropic influence on the isolated and semi-isolated heart of C. meanas (Stangier 1991; Saver and Wilkens 1992; McMahon and Wilkens 1992) and the isolated heart of the crayfish Orconectes limosus (Stangier 1991). Despite this, both the present study and that of Stangier (1991) were unable to demonstrate any significant effects of CCAP on heart rate in intact Cancer. The pericardial organs of C. maenas contain concentrations of CCAP 6.75 times greater than those of Cancer pagurus (Stangier 1991); therefore, it is possible that CCAP, like proctolin, induces species-specific effects on crustacean heart muscle and that CCAP is not a physiologically important cardiac neurohormone in the cancrid genera. This suggestion is supported by the finding that the isolated heart of O. limosus is only 35% as sensitive to 10 - 6 mol'1-1 CCAP as that of C. maenas (Stangier 1991), although data for the concentration of CCAP in crayfish pericardial organs were not presented. It is possible that CCAP may be implicated in regulation of hindgut motility in Cancer (Stangier 1991) rather than cardiovascular control. As with proctolin, CCAP infusion increased the contractility of the heart in C. magister, increasing both stroke volume and cardiac output. Sensitivity to CCAP was, however, lower than to proctolin. CCAP also had effects on arterial haemolymph flow, but in contrast to proctolin it increased rather than decreased flow through the anterolateral arteries in the majority of animals tested (Fig. 4b). Perfusion of the carapace, testes and tissues dorsal to the stomach, oesophagus and hepatopancreas was thereby increased. This increase in haemolymph delivery was enabled by an increase in cardiac stroke volume (Fig. 9) as well as possible shunting by the cardioarterial valves. Scaphognathite rate increased slightly (approximately 20%) at the higher concentrations of CCAP tested; however, this was much less dramatic than the 300% increase found in intact C. pagurus treated with 10- 7 tool. 1-1 CCAP (Stangier 1991). Infusion of either proctolin or CCAP into the pericardial sinus is associated with both increased cardiac output and alteration of the percentage of cardiac output entering specific arterial systems (Figs. 7, 8). Since each arterial system supplies particular regions of the body, peptide infusion alters haemolymph supply to these various organs. Both peptides are present in the pericardial organs and it is possible that they act as modulators of cardiac and circulatory function during exposure to environmental perturbations such as salinity, oxygen and temperature changes. The responses to proctolin infusion closely resemble the cardiovascular responses of C. magister to acute hypoxic exposure (Airriess and McMahon 1994). Crabs exposed to hypoxic conditions exhibited bradycardia and increased cardiac stroke volume for the duration of the exposure period. There was a marked decrease in haemolymph flow through the anterolateral arteries in conjunction with a doubling of flow through the sternal artery. Haemolymph flow through the posterior aorta declined initially, but increased as the severity of hypoxia increased. Somewhat similar responses to both proctolin and hypoxia have been reported for the lobster H. americanus and the cray-
fish Procambarus clarkii (McMahon and Reiber 1991; Reiber et al. 1992) suggesting that these responses may occur widely in decapod crustaceans. The redistribution of haemolymph caused by CCAP has some similarities to changes invokved by temperature variation: increased haemolymph flow occurs in the anterolateral arteries of C. magister exposed to an increase in ambient temperature (DeWachter and McMahon 1992). These workers also report a change in heart rate which was not observed in the present study but which may have been a result of either the direct effect of temperature on the burst frequency of the cardiac ganglion or the actions of other cardioactive substances (Airriess and McMahon 1992). The exact role of CCAP in environmental stress responses remains to be elucidated. In C. maenas, the release of less than 1% of the CCAP reserves stored in the pericardial organs would be sufficient to effect cardiovascular changes (Stangier et al. 1987), whereas a substantially greater percentage of proctolin reserves would have to be released to effect similar adjustments (Stangier et al. 1986). Future studies will involve measurement of circulating concentrations of these hormones to determine whether environmental perturbation causes their release into the haemolymph at levels sufficient to effect the changes in cardiovascular function reported here. Acknowledgements. This study was supported by NSERC Grant No A5762 to BRM, N S E R C and A H F M R Postgraduate Fellowships to CNA and grants from WCUMBS (Bamfield Marine Station) to IJM and CNA.
References Airriess CN, McMahon BR (1992) Aminergic modulation of circulatory performance in the crab, Cancer magister. In: Hill RB et al (eds) Comparative Physiology, vol. I I. Karger; Basel, pp 123-131 Airriess CN, McMahon BR (1994) Cardiovascular adaptations enhance tolerance of environmental hypoxia in the crab Cancer magister. J Exp Biol (in press) Alexandrowicz JS (1932) The innervation of the heart of the Crustacea. 1. Decapoda. Q J Microscop Sci 75:181-249 Alexandrowicz JS (1953) Nervous organs in the pericardial cavity of the decapod Crustacea. J Mar Biol Assoc U K 31:563-580 Alexandrowicz JS, Carlisle DB (1953) Some experiments on the function of the pericardial organs in Crustacea. J Mar Biol Assoc U K 32:175-192 Benson JA, Sullivan RE, Watson W H lII, Augustine GJJ (1981) The neuropeptide proctolin acts directly on Limulus cardiac muscle to increase the amplitude of contraction. Brain Res 213 : 449454 Bishop CA, Krouse ME, Wine JJ (1991) Peptide potentiation of calcium channel activity can be seasonally variable. J Exp Biol 156:607 610 Breidbach O, Dircksen H (1991) Crustacean cardioactive peptideimmunoreactive neurons in the ventral nerve cord and the brain of the meal beetle Tenebrio molitor during postembyronic development. Cell Tissue Res 265:129-144 Cooke IM, Sullivan RE (1982) Hormones and neurosecretion. In: Atwood HL, Sandeman DC (eds) The biology of Crustacea, vol. 3. Academic Press, New York, pp 205-290 Cumberlidge N, Uglow R F (1977) Heart and scaphognathite activity in the shore crab Carcinus maenas (L.). J Exp Mar Biol Ecol 28 : 87-107
I.J. McGaw et al. : Peptidergic modulation of cardiovascular dynamics in a crab De Wachter B, McMahon BR (1992) Temperature effects on the circulation of Cancer magister. Am Zool 32: 50A Dircksen H, Muller A, Keller R (1991) Crustacean cardioactive peptide in the nervous system of the locust, Locusta migratoria: an immunocytochemical study on the ventral nerve cord and peripheral innervation. Cell Tissue Res 263:439-457 Florey E, Rathmayer M (1978) The effects of octopamine and other amines on the heart and on neuromuscular transmission in decapod crustaceans: further evidence for a role as neurohormone. Comp Biochem Physiol 61 C: 229-237 Groome JR, Watson WH III (1989) Second-messenger systems underlying amine and peptide actions on cardiac muscle in the horseshoe crab Limulus polyphemus. J Exp Biol 145:419-437 Hartley CJ, Cole JS (1974) An ultrasonic pulsed Doppler system for measuring blood flow in small vessels. J Appl Physiol 37: 626-629 Kuramoto T, Ebara A (1984) Neurohormonal modulation of the cardiac outflow through the cardioarterial valve in the lobster. J Exp Biol 111:123-130 Maynard DM (1960) Circulation and heart function. In: Waterman TH (ed) The physiology of Crustacea, vol 1. Academic Press, New York, pp 161-226 McLaughlin PA (1983) Internal Anatomy. In: Mantel L (ed) Internal anatomy and physiological regulation. Academic Press, New York, pp 1-52 McMahon BR (1992) Factors controlling the distribution of cardiac output in decapod crustaceans. In: Hill RB (ed) Comparative Physiology, vol. 11. Karger, Basel, pp 51-61 McMahon BR, Burnett LE (1990) The crustacean open circulatory system: a reexamination. Physiol Zool 63:35-71 McMahon BR, Reiber C (1991) Effects of proctolin in controlling the distribution of cardiac output in the lobster. FASEB J 5:3390A McMahon BR, Wilkens JL (1972) Simultaneous apnoea and bradycardia in the lobster Hornarus americanus. Can J Zool 50:165 170 Mercier AJ, Wilkens JL (1985) Modulatory effects of proctolin on a crab ventilatory muscle. J Neurobiol 16: 401-408 Morris S, McMahon BR (1989) Potentiation of hemocyanin oxygen affinity by catecholamines in the crab Cancer magister: a specific effect of dopamine. Physiol Zool 62:654-667 Orchard I, Belanger JH, Lange AB (1989) Proctolin: a review with emphasis on insects. J Neurobiol 20 :470-496 Pasztor VM, MacMillan DL (1990) The actions of proctolin, octopamine and serotonin on crustacean proprioceptors show species and neurone specificity. J Exp Biol 152:485-504 Pearson J (1908) Cancer (the edible crab). Proc Trans Liverpool Biol Soc 22:291-499 Reiber CL, McMahon BR, Burggren WW (1992) Redistribution
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of cardiac output in response to hypoxia: a comparison of the freshwater crayfish, Procambarus clarkii, and the lobster, Hornarus americanus. In: Hill RB et al (eds) Comparative physiology, vol. 11. Karger, Basel, pp 22-28 Saver MA, Wilkens JL (1992) The effects of neurohormones on cardiac performance as measured by EMG and contractility in the shore crab Carcinus maenas, Am Zool 32:64A Schwarz TL, Lee GMH, Siwicki KK, Standaert DG, Kravitz EA (1984) Proctolin in the lobster: the distribution, release, and chemical characterization of a likely neurohormone. J Neurosci 4:1300-1311 Stangier J (1991) Biological effects of crustacean cardioactive peptide (CCAP), a putative neurohormone/neurotransmitterfrom crustacean pericardial organs. In: Florey E, Stefanoe GE (eds) Comparative aspects of neuropeptide function. Manchester University Press, UK, pp 201-210 Stangier J, Dircksen H, Keller R (1986) Identification and immunocytochemical localization of proctolin in pericardial organs of the shore crab Carcinus maenas. Peptides 7 : 67-72 Stangier J, Hilbich C, Beyreuther K, Keller R (1987) Unusual cardioactive peptide (CCAP) from the pericardial organs of the shore crab Carcinus rnaenas. Proc Natl Acad Sci USA 84: 575579 Stangier J, Hilbich C, Keller R (1989) Occurence of crustacean cardioactive peptide (CCAP) in the nervous system of an insect Locusta migratoria. J Comp Physiol B 154:5-11 Starratt AN, Brown BE (1975) Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects. Life Sci 17:12531256 Sullivan RE (1978) Stimulus coupled 3H-serotonin release from identified neurosecretory fibers in the spiny lobster, Panulirus interruptus. Life Sci 22:1429-1438 Sullivan RE (1979) A proctolin-like peptide in crab perieardial organs. J Exp Zool 210:543-552 Watson WH III, Augustine GJ, Benson JA, Sullivan RE (1983) Proctolin and an endogenous proctolin-like peptide enhance the contractility o f Limulus heart. J Exp Biol 103:55 73 Wilkens JL, McMahon BR (1992) Intrinsic properties and extrinsic neurohormonal control of crab cardiac hemodynamics. Experentia 48 : 827-834 Wilkens JL, Mercier AJ, Aramant R, Leah TM (1983) Neurohormonal modulation of crustacean ventilatory and cardiac rhythms. Soc Neurosci 9:1210 Wilkens JL, Mercier AJ, Evans J (1985) Cardiac and ventilatory responses to stress and the neurohormonal modulators by the shore crab, Carcinus maenas. Comp Biochem Physiol 82 C: 337343 Zar JH (1974) Biostatistical analysis. Prentice-Hall, New Jersey, USA, 718pp
