Pacific Science (1982), vol. 36, no. 3 © 1983 by the University of Hawaii Press. All rights reserved

Vascular Resistance in the Isolated Gills of macropus and pompilius1

JAMES R. REDMOND2 and GEORGE B. BOURNE3

ABSTRACT: The gills ofcephalopods represent a potential site ofregulation of vascular peripheral resistance. Measurements of pressure-flow relationships in the isolated gills ofOctopus macropus and Nautilus pompilius gave no evidence of autoregulation. Perfusion with putative neurotransmitters showed 5-hydroxy­ tryptamine, and possibly dopamine, to reduce vascular resistance. Physiological concentrations of acetylcholine and noradrenaline did not alter resistance to flow.

THE VASCULAR SYSTEM parallels exists in isolated vascular beds perfused by a the vertebrate vascular system in many of its Newtonian fluid (Green, Rapela, and Conrad morphological features. Johansen and Martin 1963). (1962) suggested that the cephalopod system During the RjV Alpha Helix expedition to could be separated into seven functional ele­ the , we examined the patterns of ments that have counterparts in the vertebrate pressure-flow relationships in isolated pre­ system. Included in these elements are the parations ofgills from Octopus macropus and resistance vessels, composed of the termina­ Nautilus pcmpilius to gain insight into what tion of the arterial system, and the exchange physical factors are important in peripheral vessels, made up ofthe capillaries and sinuses. resistance control. We also investigated the Collectively, these comprise the microvascu­ effects of some putative cephalopod neuro­ lature, and there appear to be many differences transmitters on the pressure-flow relation­ between the structures of the cephalopod ships in these isolated gill preparations. and vertebrate microvasculature (Barber and Graziadei 1965, Kawaguti 1970). In the vertebrates, changes in vascular peripheral MATERIALS AND METHODS (hydraulic) resistance at the level of the microvasculature (mostly the arterioles) are Freshly caught Octopus macropus and of paramount importance in the control of Nautilus pompilius were placed in large regional as well as overall blood flow. A cen­ seawater aquariums and maintained for at tral question, then, is: To what extent does least 24 hr before use. The afferent ctenidial the function of the cephalopod microvas­ vessel of a gill was canulated in situ after culature differ from that of the vertebrates? lightly anesthetizing the specimen with 3% This question can be answered in part by ethanol (Octopus) or 2% urethane (Nautilus) examining pressure-flow relationships and in seawater. The polyethylene tubing (PE 100, the concomitant peripheral resistance that 0.86 mm 1.0.) used as the catheter was con­ nected via rubber tubing passed through a peristaltic pump to a 250-ml glass container. I This research was conducted as part of the Alpha Helix Cephalopod Expedition to the Republic of the Canulated gills were removed from the Philippines, supported by National Science Foundation specimen and placed in seawater in a circular grant PCM 77-16269 to J. Arnold. plastic container 20 cm in diameter. A central 2 Iowa State University, Department of Zoology, Ames, Iowa 50011. drain with a vertical glass tube kept the water 3 University of Calgary, Department of Biology, level constant at 3 cm depth. This water was Calgary, Alberta, Canada T2N IN4. continuously aerated. 297

,,-,,.,.,,ruOT' .,.",-.'" "-,,..... ""'0""""', "",,,,,,,,,,,~,,,,~,,,, " ....r ....·"'...... " ... , -">.'''.'''' '<, -,'...... "._"" ,",' ,", ,~" " t -'" ~'H. , ...."",,,_- .,."...." 298 PACIFIC SCIENCE, Volume 36, July 1982

Perfusion flow rates were monitored by two Flow rates were controlled by a variable­ means simultaneously. The perfusion reser­ speed peristaltic pump (Cole-Parmer Master­ voir was suspended from a calibrated Grass flex). At a given flow rate, an increase in input FT 10 force transducer and the weight of pressure indicated an increase in vascular re­ the container was continuously plotted on sistance in the gill and, conversely, a decrease a Brush 2400 four-channel recorder. A Bio­ in input pressure signalled a drop in vascular tronix 2-mm flowthrough-type electromag­ resistance. netic flow probe powered by a Biotronix 610 The effects ofcertain possible neurohumors flowmeter, was also placed in the perfusion on vascular resistance were examined by per­ line near the gill. The output of the Biotronix fusing these substances through the gills at a 610 flowmeter was routed to a second channel constant flow rate and noting any changes in of the Brush recorder. At the low flow rates input pressures. Solutions ofthese substances used in this study, the change in weight of were prepared in millipore filtered seawater. the reservoir proved to be the most sensitive Pressure-flow experiments involved five measure of flow. Millipore filtered seawater Octopus macropus (wet wt 355 ± 73.4 g) and was selected as a perfusion fluid for two six Nautilus pompilius (wet wt 634 ± 168.8 g). reasons: (I) adequate volumes ofoctopus and Drug experiments were performed on five nautilus blood were not available, and (2) sea­ Octopus (wet wt 249 ± 85.3 g) and eight water behaves as a Newtonian fluid. Prelimi­ Nautilus (wet wt 574 ± 131.9 g). All experi­ nary viscosity studies indicate that octopus ments were conducted at 25 ± 1°C. All mea­ blood, with considerable amounts ofdissolved sures of variability are expressed as standard protein, does not behave as a Newtonian fluid. deviations. It is likely that this is true of nautilus blood as well. Input pressures to the gill were followed with a Statham P23 Db pressure transducer RESULTS connected by a T-tube to the perfusion canula near the inlet to the gill. This transducer At a constant perfusion flow rate the pres­ was calibrated daily against a distilled water sure in each gill tested varied with time in a manometer. Pressure measurements were characteristic manner. At the inception ofper­ recorded on a third channel of the Brush fusion the hydraulic resistance was relatively recorder. low and, consequently, a low input pressure

/20

~IOO X ...E 80

III 60 c ~ 10 40 IIa III C 20 ~ 0L.-_-"'__....a..__..L-_---IL....-_-'-__....L..__...... _-"'__...L._--' /0 /6 20 26 30 40 60 M/ MUTES

FIGURE 1. Vascular resistance as a function of time in an excised gill from Octopus macropus. Perfusion rate was constant at 8.6 ml·min-'. Resistance in Isolated Cephalopod Gills-REDMOND AND BOURNE 299

o 60 60 C'l :t I.. 50 50 • 40 f.~· 40 III oo.. 0; ~ 30 30

lD o 0 ~o lD 20 l/ 20 ~_. III II: 10 I Octopus 10 . •• ,.....------. Nautilus 0. ..;:-

o 2 4 6 B 10 12 14 16 IB o 2 ... 6 S 10 12 14 16 IB 20 FLOW, ml'mln-'

FIGURE 2. Pressure-flow relationship in the excised gill of two Octopus macropus (#2 and 4) and two Nautilus pompilius (# 1 and 5).

appeared. The pressure then rose, peaked at the amount of blood that would be expected a moderately high value, and then dropped to pass through each gill at normal perfusion again but not as low as the original value. This pressures. These values are 7.82 mI· min- t for reduction in vascular resistance was short­ a pressure difference across tqe gill of 5 cm lived and pressure again gradually increased, H 2 0 in a 355-g octopus and 0.79 mI· min-I often reaching high values before stabilizing for a pressure difference of 2 cm H 2 0 in a at about 30 min (Figure 1). The preparation 634-g nautilus. The magnitude offlow for the then showed a very slight but ever-increasing in vitro situation is clearly much below the resistance over the next 2 hr. After this in­ expected in vivo flow rates for the octopus gill crease, there was a rapid deterioration of the but in the same range as the expected value for preparation characterized by rapidly increas­ the nautilus gill. ing hydraulic resistance. Absolute pressures Furthermore, the pressure-flow profiles in varied considerably among specimens and in­ Figure 2 demonstrate that vascular beds in put pressures were, ofcourse, higher at higher both the octopus and nautilus gills are quite flow rates. The highest pressures (indicating distensible. For example, for nautilus # 1 gill highest resistance) occurred in octopus gills. there was better than a twofold decrease in The same pattern of events occurred in the peripheral resistance (where peripheral resis­ nautilus gills, but the pressures that developed tance, PR = Pressure/Flow) over an 18-cm were not as great. H 2 0 pressure range. The octopus gill behaved After reaching the stable point, flow rates in like manner; in octopus gill # 4 peripheral were varied and the corresponding input pres­ resistance was almost halved over a linear

sures measured. Over the range of pressures increase in pressure of 20 cm H 2 0. and flows used in these experiments the In addition to developing greater vascular pressure-flow relationship was almost linear resistance during perfusion, the octopus gill rather than the more common curvilinear differed from the nautilus gill in that the for­ one (Figure 2). This relationship varied mer developed a severe edema in the region greatly among the octopus gills but averaged adjacent to the branchial gland (i.e., the at­ t 0.32 ± 0.14 mi· min- . cm H 2 0 (range: tached edge ofthe gill). The most likely reason 0.03-0.76). Variation was not as great in the for the edema was a high filtration rate of nautilus gills and the corresponding average water from the branches of the afferent vessel t was 0.45 ± 0.09 mi· min- • cm H 2 0 (range: that form part of the blood supply to the 0.13-0.71). Using Fick estimates of cardiac branchial gland. output published by Johansen, Redmond, Since the octopus gill normally operates and Bourne (1978) and Wells (1979) and as­ with a small back-pressure between it and the suming that blood is equally distributed to heart (Johansen and Martin 1962), the effect

each gill, an approximation can be made of of a 2-cm H 2 0 back-pressure applied to the

., _,. _ ,,'''~,. ,.",,'" ... ~_~., ,... , ••Tt" .. "" .. ",.,,, n, " ...,r~ ...",""" ,,,.,.,... ,,. , •• ,.,..,.-,,,,,._,~•• ~ _"...... "N,..-...... ~',',"" ... • .. • 300 PACIFIC SCIENCE, Volume 36, July 1982

o 40 {II I E 30 u 30 W w ~W 0:: 0 :J o-~ (/) 20 20 -9 (/) V'0 W -9 0:: Q. 10 -6 10

Octopus Nautilus

o 11-'---1__-1-_---1__...1 50 100 100 '50

MINUTES

FIGURE 3. The effect of 5-hydroxytryptamine on the vascular resistance of excised gills of Octopus macropus and Nautilus pompilius. The upper left insert is a reduced drawing of the recorded response of the Octopus gill. Negative numbers indicate concentrations of 5-HT (e.g., -6 = 10-6 M); W represents perfusion with filtered seawater only. Perfusion rates: Octopus, 3.8 ml·min-' ; Nautilus, 5.5 ml·min-' .

efferent ctenidial vessel was tested. This had (Figure 3). At 10- 5 M, the effect on the nau­ no apparent effect on the high vascular resis­ tilus gill was strikingly reversed, with resis­ tance measured in the octopus gill. tance sharply increasing (Figure 4). As often Gills were perfused with some putative ce­ happens in drug tests, it frequently was not phalopod neurohumors dissolved in seawater possible to reverse completely the effects of in order to gain possible insights into control an application of a drug by washing. Conse­ of gill vascular resistance. Concentrations of quently, the results are complicated by the 10-8 to 10- 5 M acetylcholine did not alter the effects of one or more previous drug appli­ resistance of the octopus gill nor did 10- 8 M cations influencing the response to a succeed­ concentration affect resistance of the nautilus ing application. gill. At 10-3 M, acetylcholine caused the oc­ Preliminary results oftwo experiments with topus gill to contract, increasing resistance to dopamine indicate that this substance, at flow. This is believed to be a pharmacological 10- 8 -10-6 M, may also reduce vascular resis­ response to abnormally high concentrations tance in the nautilus gill, although the effect of acetylcholine rather than a physiological does not seem as great as that of 5-HT. response. Noradrenaline at 10- 6 M similarly had no effect upon resistance of the nautilus gill; it was not tested in the octopus. DISCUSSION 5-Hydroxytryptamine (5-HT), on the other hand, had a marked effect on vascular resis­ One recurrent problem throughout these tance of the gills of both . In Nautilus, experiments, due in part to our limited equip­ 10- 9 M 5-HT clearly reduced gill vascular ment aboard ship, was our inability to main­ resistance. Higher concentrations (10- 6 M) tain a preparation for a long enough period to were required for a similar effect in Octopus permit repeated pressure-flow profiles. Addi-

«-"'" >T > ,~, - .. >" , ~,- _. """ ,,, ~ " 1_ ... •• < -" " , 10" '" , , _. - Resistance in Isolated Cephalopod Gills-REDMOND AND BOURNE 301

40 accounted for on anatomical grounds. In 0 nautilus there is no branchial gland running ~ along the dorsal edge of the gill as there is in :I -5 octopus. The circulation arrangement in the 30 ° E branchial glands ofoctopus is peculiar in that u -5 . 0 these glands receive blood from branches of W afferent branchial vessels as well as from ar­ a:: 20 w I. :J '~O/ teries arising from the aorta. Blood is drained lD -9\ from the branchial glands by veins leading to lD ~ -7 the lateral venae cavae (Isgrove 1909). The W W a:: 10 %W edema and higher vascular resistance of the Il. We' -IS octopus gill was probably due to either drain­ age or filtration ofperfusate from the afferent branches into the branchial gland. o y' J!__L-_--'-__..L-_--I.__.... 100 150 200 The basic relationship between pressure and flow in both nautilus and octopus gills MINUTES was a linear one rather than the curvilinear FIGURE 4. The effect of 10- 5 M 5-hydroxytryptamine relationship observed by many mammalian on the vascular resistance of an excised gill of Nautilus workers (see Green et al. 1963). A curvilinear pompilius. Negative numbers indicate concentrations of relationship between pressure and flow has 5-HT (e.g., - 5 = 10- 5 M); W represents perfusion with filtered seawater only. Perfusion rate: 5.7 ml·min-'. been described by Bourne (1979) for part of the systemic vasculature in . Mam­ malian workers have ascribed the curvilinear tionally, poor viability of the gills could have relationship to passive distensibility in "non­ been caused by the use of filtered seawater as reactive" or "passive" tissues, i.e., vascular the perfusate. As explained previously, our beds that do not show autoregulation ofvaso­ choice of this perfusate was prompted by a motor tone (Green et al. 1963, Kuida 1965). desire to use a Newtonian fluid that would The linear relationship seen here appears to clear our results of complications caused by indicate that the isolated octopus and nautilus any anomalous viscosity characteristics of gills could be included in the category of pas­ cephalopod blood. sive tissues. The other type of vascular bed, The initial low peripheral resistance at the the autoregulating kind, typically shows sig­ start ofperfusion could have been caused by a moid pressure-flow relationships, a situation locally triggered vasodilation in response to seen in some fish gills (Wood 1974) but not in hypoxia or to the anesthetic used during canu­ any ofour preparations. lation. Although no actual measurements Our experimental arrangement did not per­ were made of oxygen tension, the manner of mit us to look for a critical closing pressure preparation ofthe isolated gills was such that in the cephalopod gills. This pressure is one the gills could conceivably become hypoxic below which flow would not occur because of for a short period prior to perfusion. The collapse of the vascular channels (Nichols et origin ofthe slight vasodilation prior to stabi­ al. 1951). lization could not be ascertained. The increas­ The chemical agents used (acetylcholine, ing vascular resistance that characterized the dopamine, 5-HT, and noradrenaline) are sus­ decay ofthe preparation has been observed in pected neurotransmitters of cephalopod cen­ squid systemic vascular beds (Bourne 1979) tral nervous systems (see Tansey 1979 for a and in fish gills (e.g., Keys 1931, Wood 1974). review ofcephalopod neurotransmitters). The It might simply represent rigor contractions action of these substances on isolated mol­ of the vasomotor systems as the preparations luscan hearts is well known (Hill and Welsh lose their viability. 1966). Much less is known about the role of The difference in behavior of the nautilus these putative neurotransmitters in the rest and octopus gills during perfusion can be of the circulatory system. Although acetyl- 302 PACIFIC SCIENCE, Volume 36, July 1982

choline appears to have no physiological action role in controlling different aspects ofgill func­ on the isolated gill of nautilus and octopus, tion in the three major classes of mollusks, it does cause a dose-dependent vasodilation but more study is needed before a full under­ in the isolated, posterior systemic circulation standing of this role is possible. ofsquid (Bourne 1979). Johansen and Huston The other putative cephalopod neurotrans­ (1962) found that acetylcholine acted as a mitter that showed vasodilatory activity in the peripheral vasodilator in intact Octopus nautilus gill was dopamine. Time limitations dofleini but with less potency than noradren­ prevented testing in the octopus gill. The ap­ aline. The reason that high doses of acetyl­ parent difference in sensitivity of the nautilus choline (10- 3 M) cause contraction and gill to 5-HT and dopamine might simply be increased peripheral resistance ofoctopus gills caused by the much higher rate of autoxida­ is not known at the present time. tion of dopamine, thus obscuring its effective Like acetylcholine, noradrenaline had no dose. It is generally known that dopamine is effect on the peripheral resistance in the iso­ especially sensitive to autoxidation in alkaline lated nautilus gill. This lack of gill vascular media. Thus, while the isolated gills of oc­ responsiveness to noradrenaline seems to be topus and nautilus do not show evidence of a major difference between cephalopod gills autoregulation, it appears likely that nervous and fish gills. In isolated fish gills, the cate­ control of gill vascular resistance exists in the cholamines, primarily adrenaline, cause an intact . a-vasoconstrictor effect superimposed on a The fact that both dopamine and 5-HT pro­ more dominant fJ-vasodilator effect (Payan duced the same action in the nautilus gill has and Girard 1977). parallels in the bivalve, Mytilus edulis, where Of the four putative neurotransmitters, 5­ exogenous 5-HT and dopamine both stimu­ HT caused the greatest response in the cepha­ late frontal ciliary activity ofthe gill (Malanga lopod gill and thus is a candidate for a role in 1975). Dopamine is also known to be active in normal vasomotor control. This assessment is the gill of the gastropod, Aplysia, where it based on the relatively low concentration of causes asynchronous movements of efferent 10-9 M 5-HT that caused vasodilation in the vessel trunklets, pinnate longitudinal muscles, nautilus gill. To date, the major role assigned and afferent vessels (Swann, Sinback, and to 5-HT in the cephalopod cardiovascular Carpenter 1978). In the nautilus gill it is system is one ofcardioacceleration and stimu­ possible that 5-HT and dopamine might be lation of cardiac contractility (Johansen and acting at a composite dopaminej5-HT recep­ Huston 1962). However, the vasodilatory tor. This type of receptor was first postulated effect on the gill is not surprising because the by Woodruff (1971) to account for dopamine same factors that would call for cardiac aug­ and 5-HT activity in certain cells of the Helix mentation would necessitate increased blood aspersa brain. However, more research is flow through the gills; hence, the lower gill necessary before we can accept a composite peripheral resistance. 5-Hydroxytryptamine dopaminej5-HT receptor as the active site has been found in the gills of other mollusks. causing the similarity of action of dopamine In the gastropod, Aplysia, 5-HT has been lo­ and 5-HT in the nautilus gill. calized in the gill (Perez and Estes 1974) and This study ofpressure-flow relationships in has been shown to stimulate cyclic adenosine­ isolated gills of nautilus and octopus lends 3', 5'-monophosphate (cyclicAMP) produc­ further weight to the contention by Bourne, tion in slices of gill (Kebabian, Kebabian, Redmond, and Johansen (1978) that the and Carpenter 1979). The physiological role nautilus cardiovascular system is functionally of 5-HT in Aplysia gill is not understood more similar to the coleoid system than it is (Kebabian et al. 1979). In bivalves, e.g., different. The few differences that were ob­ Mytilus and Modiolus, 5-HT has been shown served appear to have an anatomical basis. to stimulate particle transport by cilia Although the linear pressure-flow relation­ (Gosselin and O'Hara 1961). These studies ships ofthe cephalopod gills are analogous to indicate that 5-HT might have a physiological those of fish gills, the cephalopod gills show

~ , ~ • • ,. L' ,. '" ,.., "" •• , _". ." ,, ~ , ". ,_ ," , Resistance in Isolated Cephalopod Gills-REDMOND AND BOURNE 303

clear molluscan affinities in the types ofputa­ KAWAGUTI, S. 1970. Electron microscopy on tive neurotransmitters that have vasomotor muscle fibers in blood vessels and capil­ activity. lariesofcephalopods. Okayama Univ. BioI. J.16:19-28. KEBABIAN, P. R., J. W. KEBABIAN, and D. O. CARPENTER. 1979. Regulation of cyclic LITERATURE CITED AMP in heart and gill of Aplysia by the putative neurotransmitters dopamine and BARBER, V. c., and P. GRAZIADEI. 1965. The serotonin. Life Sci. 24: 1757-1764. fine structure of cephalopod blood vessels. KEYS, A. B. 1931. The heart-gill preparation I. Some smaller peripheral vessels. Zeit. of the eel and its perfusion for the study of Zellforsch. Mikrosk. Anat. 66: 765-781. a natural membrane in situ. Zeit. Vergl. BOURNE, G. B. 1979. Pressure-flow relations Physiol. 15: 352-363. in the perfused systemic circulation of KUIDA, H. 1965. General relations ofpressure squid. BioI. Bull. 157: 358-359 (Abstr.). and flow. Ann. N.Y. Acad. Sci. 127: 364­ BOURNE, G. B., J. R. REDMOND, and 372. K. JOHANSEN. 1978. Some aspects ofhemo­ MALANGA, C. J. 1975. Dopaminergic stimu­ dynamics in Nautilus pompilius. J. Exp. lation offrontal ciliary activity in the gill of Zool. 205: 63-70. Mytilus edulis. Compo Biochem. Physiol. GOSSELIN, R. E., and G. O'HARA. 1961. An 51C:25-34. unsuspected source of error in studies of NICOLS, J., F. GIRLING, W. JERRARD, E. B. particle transport by lamellibranch gill CLAXTON, and A. C. BURTON. 1951. Funda­ cilia. J. Cell Compo Physiol. 58: 1-9. mental instability ofthe small blood vessels GREEN, H. D., C. E. RAPELA, and M. C. and critical closing pressures in vascular CONRAD. 1963. Resistance (conductance) beds. Amer. J. Physiol. 164: 330-344. and capacitance phenomena in terminal PAYAN, P., and J.-P. GIRARD. 1977. Adren­ vascular beci~. Pages 935-960 in W. F. ergic receptors regulating patterns of blood Hamilton, ed. Handbook of physiology. flow through the gills of trout. Amer. Section 2: Circulation. Vol. II. American J. Physiol. 232:HI8-H23. Physiological Society, Washington, D.C. PEREZ, B., and J. ESTES. 1974. Histology and HILL, R. B., and J. H. WELSH. 1966. Heart, histochemistry of the peripheral neural circulation and blood cells. Pages 126-174 plexus in the Aplysia gill. J. Neurobiol. in K. M. Wilbur and C. M. Yonge, eds. 5:3-19. Physiology of . Vol. 2. Academic SWANN, J. W., C. N. SINBACK, and D. O. Press, New York. CARPENTER. 1978. Dopamine induced mus­ ISGROVE, A. 1909. Eledone (the octopod cut­ cular contractions and modulation of tlefish) memoirs. Liverpool Marine Biolog­ neuromuscular transmission in Aplysia. ical Committee. 18: 1-105 and plates. Brain Res. 157: 167-172. JOHANSEN, K., and M. J. HUSTON. 1962. TANSEY, E. M. 1979. Neurotransmitters in the Effects of some drugs on the circulatory cephalopod brain. Compo Biochem. Physiol. system of the intact, non-anesthetized 64C: 173-182. cephalopod, Octopus dofleini. Compo Bio­ WELLS, M. J. 1979. The heart beat of Octopus chern. Physiol. 5: 177-184. vulgaris. J. Exp. BioI. 78:87-104. JOHANSEN, K., and A. W. MARTIN. 1962. Cir­ WOOD, C. M. 1974. A critical examination of culation in the cephalopod Octopus dofleini. the physical and adrenergic factors affecting Compo Biochem. Physiol. 5: 161-176. blood flow through the gills of the rainbow JOHANSEN, K., J. R. REDMOND, and G. B. trout. J. Exp. BioI. 60:241-265. BOURNE. 1978. Respiratory exchange and WOODRUFF, G. N. 1971. Dopamine receptors: transport of oxygen in Nautilus pompilius. A review. Compo Gen. Pharmacol. 2: 435­ J. Exp. Zool. 205: 27-36. 439.

~. , ,. • - - ~ -" , -, ." , ... , , • ~... •• ~ - ~" - ..", .....>-