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A neurohormone of cephalopods with cardioexcitatory activity
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
A Neurohormone
21, 267-277 (1973)
of Cephalopods DONATELLA AND
Institute
with
Cardioexcitatory
Activity
BLANCHI, LUIGI NOVIELLO, MASSIMO LIBONATI’
of General Zoological
Physiology, University Station. Naples, Italy
Received September
of Turirc,
22. 1972
A neurohormone (NS) with long-lasting cardioexcitatory action on the isolated heart of cephalopods, has been extracted from the neurosecretory system of the vena cava (NSV system) of several cephalopod species. Electrical stimulation of the NSV nerves to the vena rava liberated a cardioexcitatory substance. Cardioexcitor extracts from Octopus vulgaris have had their activity increased 95 times by purification procedures. The resulting material is homogeneous on electrophoretic analysis, shows an absorbance maximum near 265 nm, and has a molecular weight of about 1366. NS is not identifiable with any ratecholamine or indole compound lmown to be present in cephalopod tissues.
The system of nerves which reach the anterior part of the vena cava of cephalopods, ending as a dense network of nerve fibers in close proximity with the blood stream, was first described by Alexandrowicz (1964, 1965) and named by him the neurosecretory system of the vena cava (NSV system). Axons of this system originate from nerve cells located in a layer of the palliovisceral lobe of the brain and in paired ganglionic trunks (lateral and medial NSV trunks) which emerge from the visceral lobe with the posterior infundibular and the visceral nerves, respectively. NSV nerves from these trunks 1:enetratc the muscular wall of the vena cava, forming a fine neuropile adjacent to the inner surface of the vessel. Martin (1968)) examining the fine structure of the NSV system in Octopus, demonstrated the presence of electrondense granules similar to those described in known neurosecretory tissue. Both authors suggested a neurosecretory function for the system. Blanchi (1969a,b) investigated the postu’ Present address: Laboratory of Biological Chemistry, Faculty of Sciences, Naples. 267 Copyright @ 1973 by Academic Prr::s. Inc. .4ll rights of reproduction in any form reserved.
lated secretory function and the physiological role of the NSV system with hearts isolated from various cephalopods. Extracts prepared from the innervated region of the vena cava, when added to the perfusion fluid, increased the frequency and amplitude of cardiac beat. It was suggested that the NSV system has the function of liberating a neurohormone involved in the regulntion of cardiac activity. Similar results and conclusions were presented by Berry and Cottrell (1970) after their study of fine structure of the neuropile of the vena cava of Eledone cirrosa. This paper describes further efforts to characterize and identify the active material of this neurosecretory system. In the first part a more detailed examination of function of the postulated neurohormone is described, in the second part a description of its gross chemical properties is presented. MATERIALS
AND
METHODS
Preparation of eztructs. Extracts were from the anterior innervated portion of cava of Octopus vulgaris Lam., Octopus Risso, Bledone moschata Lam., Sepia
prepared the vena mucropus officinalis
268
BLANCHI,
NOVIELLO,
L., and Loligo vulgaris Lam. as follows: a single vein was dissected from the experimental animal, cut longitudinally and immersed in 5 ml of distilled water for 15 min at room temperature (crude extract). Extracts of the abdominal aorta, prepared under identical conditions, were used as controls. The crude extracts were immediately used or stored at -30°C until use, berause at room temperature they lost activity. Bioassuy. Isolated and perfused hearts from the five species mentioned above were used for bio!ogicnl assays. Routine assays of activity of ihe neurosccretory substance, especially during the purification procedure, employed hearts from Octopus vnlgmis. The isolatrtl hearts were cannulated through the abdominal aorta and attached by the efferent branchial rcssels to a photoelectrical transduccxr directBy connected to the dc amplifier of a physiograph (Four A, E.M. Instruments Co., Inc.) ; heart recordings were isotonic. The hearts were perfused at a rate of about 20 ml/min. at constant pressure, with oxygenated sea water. Usually lo-50 ~1 of extract or of any fraction from the purification steps were diluted to 1 ml with filtered sea water and were injected into t,he perfusion system, not far from the heart: the dead time of the perfusion system was about 7 sec. In this way the injected solutions were diluted before reaching the heart and were washed out quickly thereafter. It was, therefore, not possible to determine absolute threshold. Concentrations givm for any testrd substance refer to the injected solution (1 ml) and are given in terms of units, 1 unit corresponding to the cardioexcitatory action of 10 ng of 5-hydroxytryptamine (5-HT). Spectrophotometric procedures. Absorbance at 265 nm (determined with a Zeiss PM& 11 spectrophotometer) was used to follow the active principle during the purification procc,dure or to its concrniration in test indicate, nlt,crnatively, solutions. The wavelength of 265 nm corresponds to the maximal nbsorhancr (see Fig. 9) of purified fraction of NS, as determined in pilot experiments. Chromatography. Unless otherwise specified. all ol)erations wrre carrirrl out at 0-4°C. Gel chromntograpl~y was prrformcd using Srph:&r G-75 superfine (Pharmacin Upl’snla) , pnckt~l in a K 15/90 c~olumn. 78 cm high, cctuilihrnted with 0.2 M NH,HCO,,, pH 7.S. Chromatography through Bio-Gel P-2 (Bio-Rad) was performed using bidistilled water as eluent. A flow rate of 10 ml/hr was obtained with a constant-volume pump (LKB-10200 Perspcx peristaltic pump) ; 2.5 ml fractions were collected. The Fo of the column was always determined using blue dex-
AND
LIBONATI
tran. Chromatography on CM-Sephadex C-25 (Pharmacia Uppsala) was conducted on columns equilibrated with 0.01 M Tris.HCl buffer (buffer A) at pH 8 (conductivity of 6-8 millimhos/cm). il stepwise grsdient between 0.1 and 1.0 M NaCl was used for elution (Fig. 7). Preparative chromatography was performed on thin-layer plates of Silica Gel F,,, (Merck, 20 X 20 cm) with a thickness of 0.25 mm. Development was performed one-dimensionally with nhutanol, acetic acid, and water (120:30:50) for 4 hours at, room temperature. Spots were detected by spraying with a solution of 0.1% Ce(SO,), in concentrated HSO, or with Ehrlich reagent (sometimes modified according to Smith, 1969). followed b,v heating at, 110°C for 4 min. Gel-electrophoresis. (A) Cellogel strips, 2.5 X 17 cm, were buffered in sodium Verona1 (8.24 g/liter) at pH 9.6 for about 2-3 hr (excess of buffer being rrmoved by gentle pressure with a filter paper sheet), and electrophorcsis was carried out at 200V for 75 min. Strips were then stained in an Astral blue solution (1% in 1% acetic acid; Beccari and Mazzi. 1966) for 12 hr and destained with SC% acetic acid. (B) Cylindrical polyacrylamide gels (Ornstein. 1961), 5% w/v, 6.5 mm in diameter, were used. Samples of the most purified fraction (NS,, Table 1. pool of several preparations), 50 or 100 ~1 in volume. corresponding to 0.05 or 0.1 OD units at 265 nm, rrsprclivrly, were mixed with equal volumes of 5O’i (w,‘v) glycerol and layered on the top of grls. Electrophoresis was carried out with a constant current of 5 V/cm per tube, at room t,rmperature. for about. 60 min. The buffers used were 0.05 Jf tris.glycine, pH 9.2, or palanine buffer of Reisfeld et al. (1962). Gels were stained with Ast,ral blue for 12 hr and destained in 5% acetic acid for 24 hr. Other nr,e&~Z.s. The following analytical methods were used: for carbohydrates the method of Hodge and Hofreiter (1962); for amino nitrogen that of Moore and Stein (1948). Proteins were determined by ihe 280:260 nm absorption quotient (Warhurg and Christian, 1942) or by the microbiurct method (Bailey, 1962). The method of Warren for sialic acid (1959), and that of Elson and Morgan (1933) for hexosamines were usrd. The ionic pattern of the CM-Sephadex C-25 chromatography was determined with a conductivity meter, t,ype CDM2 (Radiometrr. Copenhagen). Chemicals. Ninhydrin and acetylacetone were from Merck. Other chemicals from Sigma Chem. Co. Pronasc ~vas purrlrnsed from Koch-Light Lab. Ltd.: Astral blur from Gurr’s Ltd., London. England.
CEPHALOPOD
CARDIOEXCITOR
NEUROHORMONE
t NS
269
t
I 1 min
t 5-HT FIG. 1. Isolated heart of Octopus vu[garis; arrows indicate NS (absorbance at. 265 nm, 0.436) and 5-hydroxytryptamine,
RESULTS
Biological Characterization of the Vena Cava Extracts of Octopus vulgaris The remarkable cardioexcitatory action of the extracts of the vena cava-innervated region consists of a long-lasting positive inotropic and chronotropic effect. The rtsponse grows to a maximlim 30-45 set after the injection and then persists for as long as 20 min (Fig. 1). With higher concentrations of extracts an increase in tone can be observed, which finally leads to systolic
t 10
'
125
Fro. 2. Action of heated NS and indicate application of test solutions. of NS corresponds to an absorbance Records were cut and rearranged 2030 min. Note that the time from to first. noticeable response is longer
'
150
application of tesl. solubons: 500 ng. Vertical calibration
heated : 10 g.
(see text)
arrest. After washing, the heart starts beating again. Repeated introduction of the extract results in a progressive reduction of the response. Control experiments were carried out by injecting aorta extracts; in this case no variation of the beating was observed. The action of Octopus vulgaris NS was compared with that of epinephrinr, norepinephine, and 5-hydroxytryptamine : these substances have a marked positive inotropic and chronotropic action on the heart of O&opus, but the effect is transient,
I
I100
'
1200
of 5-hydroxytryptamine (5-HT) on the isolat.ed Oclopus heart. Arrows Indicated NS amounts are expressed in microliters of extract: 1 ~1 of 1.09 X lOa3 at 265 nm. 5-HT amounts are given in nanograms. to facilitate dose-response comparisons: at X, record was stopped foi injection to maximum response is approximately constant, though time for more dilute injections.
“70
t!I,.\h-CHI,
NOVIELLO,
AND
LIBONATI
t cw 1 min VW. 3. Action heart of 0ckq1u.s
of 50 ~1 of “uJllt,rol water’J (CR) and “stimulat~iori At S, record was stopped for 20-30 min.
lasting not more than 2 min. The acccleration obtained with 5-hydroxytryptnmine was often followed, during washing, by a period of depressed activity. In Fig. 2 the action of NS is compared to that of 5-HT. Electrical
water”
(hw)
isee lest)
on the isolaled
uulgaris.
Xtimulation
Expehnlents
The vena cava, the medial ncurosecretory nerves (NW nerves) with the visceral nerves were carefully dissected out and put in 5 ml of oxygenated sea water; SSV nerves were picked up on silver electrodes and stimulated with trains of pulses every 10 see, 1 WC duration, at a frequency of 100 Hz; each pulse with a duration of 0.5 millisec and an amplitude of 2-5 V. After stimulation for 15, 30, and GOmin the bathing medium (“stimulation wakr”) was assayed on the heart. (‘ontrols con>istccl of
identical preparations without stimulation (L’control water!‘) Figure 3 shows the effect of successive injections of 50 ~1 (in 1 ml of sea water) of “control water” and of “stimulation water,” from a preparation that, had been stimulated for 30 min as dcscribed abovca. An increase of nmplituclc and frequency follows I)oth injections, but a significantly stronger effect can be observed in th(, rnsc of the “stimulation water.” With smaller closes or shorter periods of stimulation and immersion (of the vena envat “control water” has no cffeet, while “stimulation water” ohtaincd under iclent8ical conditions, has a cartlioexcitatory action qualitatively similar to that produrcd by the vrna cava extracts. The small exrit,atory effect of the ‘Lront’rol water” indicatcb:: that some NS i:: rclcasrd by the tissue n-hen uiistimulntcrl.
1 min
FIG. 4. (A) A&m of cr~tde NS of Octopus vu1gari.s (0.) and of Elrdonc moschnta (E;.) (absorbance at’ 265 nm, 0.350) on the isolated heart of Eledone. (B) Action of crude NS of Sepkx (S.) and of Octopus ~lgaris heart of Scpiu. Arrows indicat,e application of test solu(0.) (absorbance at 265 nm, 0.020) on the isolated Gons. At X, record wa.s stopped for 20-30 min. The same Octopus extract, was injected in both hearts, hlit it, was dilut,ed 17.5 times to be tested on Sepia heart.
CEPHALOPOD
Procedure >Crude ext,rtlction Itea+ Sephades G-75 Bio-Gel P-2 CRI-Rephades C-25 Thin layer (preparative)
Frac(ioll NS .;I KS, NP, h’si, ?u’S&
CARDIOEXCITOR
Volume (ml) 110.0 10s.0 4 2 1 0.
Total Imits
Yields c”; 1
723 43XS 1 SO4 740 260 1.31
100. II0 B” so 21 !I0 10 “:I :: .60 2.10
:3 0 3 1
“71
NEUROHORMOSE
Total :hsorl~ance nt 26.5 11111 550 0 Y4S.4 “8.6 1.4 0.4 0.12
Specific activity units ’ absorbance at, 265 nm 1:i. 1 1x2 76.4 525.6 650.0 1250.0
Relative pllrification
1.4 5,s 40. :I 49.6 95 .4
-
Assays of Vena Cazla Extracts on lIonor Species or on Different Species of Cephalopods
Incubation carried out with trypein or papain under the same conditions did not affect the activity of NS on the isolated Octopus heart.
Extracts were prcpartd from the venae cavae of the 5 species easily available in Purification of the Vena Cava Extract &he Bay of Naples (Octopus vulgaris, Octopus ~w~c~opus, Eledone moschata, Sepia A typical purification procedure is sumoficinalis, and Loligo vulgaris). The action marized in Table 1. Protein concentration of NS of each species was tested on the I 1 heart of the same and on that of the other -ABSORBANCE 2E5nm species: increase of frequency and ampli~.-.~ ABSORBANCE 2BiJnm tude of the beat was always observed. Figure 4 shows that the injection of crude .,extract produced a marked effect in the case of the direct and of the crossed experiments. The comparison was made on the basis of total activity per vena cava and mtasuring the absorbance at 265 mn of the injected extracts. The NS of any octopod sllecies is equally cffectivc on the hearts of other octopods, while it must bc diluted lo-20 times for similar effects on the tlecapods Sepia and Loligo. In like manner, 301 NS from the two decapod species are equally effective on the decapod hearts but must be concentrated at least ten times *to produce marked effect on octopod hearts. nigestim of Cmde Esfrncf u\ifh Pronase Incubation of 0.5 ml of crude extract with 0.2 mg of Pronase at 37°C for 2--3 hr destroyed 8&90% of the activity present ‘in the untreated extract. Control expcriments with equivalent amounts of pronase solutions in 1 ml of sea water had no effect on heart heat.
FIG. 5. Gel-chromatography through Sephades G-75. NS? preparation (absorbance at 265 nm, 126) was applied onto a 1.5 X 78 cm column and eluted at 4°C with 0.2 III NHIHCO~, at pH 7.8. Flow rate, 10 ml/hr. Active fractions (62-77) were pooled.
272
BLANCHI,
NOVIELLO,
has been taken as an index of active material concentration through the first purification steps. The “specific activity” was defined as the ratio between biological activity unit and the absorbance at 265 nm, where the active substance (partially purified in pilot experiments) showed it’s maximum. Examples presented in the figures have
46
-
36
-
26
-
16
-
6.0
AND
LIBONATI
been taken from different, individual experiments. Xtep 1. Crude extract (NS,) was prepared by the procedure described under Materials and Methods (Preparation of extracts). Step Z?. The crude extract was heated in a boiling water-bath for 15 min, and the insoluble material was removed by cen-
a
s2.0 r u ml.8 a
-
18
El.6
-
16
u 1.4
-
1.2
-
1.0
-
0.8
-
0.6
-
8
I
6
I,Y :’J: -i
40
50
60
70
80
(
FRACTIONS FIG. 6. Chr0matograph.v of N& preparation (absorbance at 265 nm, 260) through a Bio-Gel P-2 column (1.5 x 78 cm). Flow rate, 10 ml/hr; temperature, 4°C; 2.5-ml fractions were collected. Shaded area indicates fractions containing biological activity (activity units/ml X 10). Fract.ions IS-26 were pooled (NW -- m, Absorbance at 265 nm; - - -, absorbance at’ 280 nm; O-0, carbohydrat’es (&ml); . . ., amino nit,rogerl (~moles/ml).
CEPHALOPOD
CARDIOEXCITOR
tivity ; the third peak represented the active fraction (NS,). A typical experiment is shown in Fig. 5. Step 4. Fraction NS, was now lyophilized, pooled wit,h others prepared from similar experiments, dissolved in bidistilled water, and applied onto a Bio-Gel P-2 column. At least 4 peaks (absorbance at 265 nm) emerged (Fig. 6). Activity was found only in the first peak. The material was lyophilized again and dissolved in bidietihed water (XS,). Step 5. Fraction NS, was applied to a
trifugation at 27,000g for 15 min at M”C. ‘Supernatant solutions were lyophilized !NSA. The activity is unaffected by heating, a sample of heated extract being equally active as a same sample of crude extract. Step S. Preparation NS, was dissolved in >bidistilled water and applied onto a Sephadex G-75 ‘Superfine” column. The effluent showed three peaks, absorbing at 280-260 nm ; t,wo of them, corresponding to about 90% of the total protein input., were exeluded since they were devoid of any acbufferA
bufferA+ 0.1 M NaCl
50ml
50ml
273
NEUROHORMONE
buffer A + 0.5 M Na C I
buffer A+ 1.0M Na Cl
100ml
1.4 -
I 55
1.3 .*I’
I:.
...:’,.*.4.. . .. . ._.. . . . . . .
1.2 -
1
1.0 -
40
:..“.
. . . ..a...
. . . . .
z-
..-.
0.9 -
,+; :: ::: .
z 0.6 z =: 0.7 cp u
50
45
, ,
1.1 -
w v
-1
100ml
:
; :
-35
,c a
-30.
& m
-25
z
::
i
:: :.i
:
;
0.6 0.5 0.4 0.3 0.2 0.1
25
50
75
100
125
150
175
200
225
250
275
ml of eluate FIG. 7. Chromatography of NS, preparation (absorbance at 265 nm, 18.4) through a CM-Sephadex C-25 column (1 X 10 em). Elution was carried out by stepwise gradient using sodium chloride in 0.01 M trisHC1 buffer (buffer A), pH 8; 2.5-ml fractions were collected. Flow rate, 10 ml/hr; temperature, 4°C. Shaded area indicates active fractions (between 2.5 and 25 ml of eluent) (activity units/ml X 10). --, Absorbance at:265 nm: - - -, absorbance at. 280 nm; . . . ., conductivit)y (mmhozcm).
274
BLANCHI,
NOVIELLO,
CM-Sephadex C-25 column and eluted with a stepwise gradient (Fig. 7). Activity was found in the first 30 ml of eluate. The active fractions were pooled and lyophilized (NS,) . Step 6. Preparative chromatography on thin-layer plates. Fraction NS, was applied on a Silica Gel F,,, plate (see Materials and Methods). Two bands were detected; only one of them, with a lower Rf, was active (NS,) . Criteria
AND
LIBONATI
TABLE
2
DETERMINATION OF THE MOLECULAR WEIGHT OF PUKIFIED NS ACCORDING TO ANDREWS (1965)= Substances Riboflavin Coenxyme A NS Cyanocobalamin
Elution volume
Molecular weight
120.7 68.0 47.6 42.5
376.4 767.6 1280.0 1355.4
(found
)
a Bio-Gel P-6 column, 0.9 X 90 cm, equilibrated and eluted with distilled water. Flow rate, 10 ml/hr. Fractions of 1.7 ml were collected. The column was subsequently loaded with 0.5 mg of riboflavin, 0.2 m,$ of CoA or cyanocoba.lamin and with N& (pool of several preparations; t,otal absorbance at 265 nm = 1.2). The elution pat,tern of the various substances wm det,ermined by absorbance at. the following wavelengths: 450 nm for riboflavin, 260 nm fat coenzyme A, 360 nm for cyanocobalamin, 265 nm for N&.
of Pwity
The electrophoresis pattern of the NS, preparation on Cellogel shows a single band, stained with Astral blue, even if rather large amounts of the substance arc applied. Analytical disc electrophoresis of NS,, also shows a single band (Fig. 8). Electrophoresis was carried ol7t ad described under Materials and Xfethods. Each gel was split longitlldinally: one half was Ultraviolet Absolptioti Spectrum stained with Astral blue, the other was The uv spectra of NS, dissolved in water sliced in 2-mm pieces. Each piece was then or at pH 1 show a broad maximum around homogenized in 1 ml of bidistilled water. 265 nm (Fig. 9, curves 1 and 2). At pH 11 After centrifugation at 10,OOOgfor 10 min. this maximum disappears, but a shoulder t,he supernatant was tested for biological activity under standard conditions. The appears in the region between 240 and 255 cardioexcitatory activity was found in the nm (Fig. 9, curve 3). The absorbance ratid region of the gel corresponding to t.he 280:260 nm, as measured from the spertrum, was 0.84. stained band of the counterpart. Behavior
Molecular Weight
Chromatographic
The molecular weight of the purified NS was determined by the gel-filtration m&hod described by Andrews (1965). A Bio-Gel column (see Table 2 for experimental details) was loaded with NS, and SUCCCSsively, under identical experimental conditions, with riboflavin, coenzyme A, and ryanocobalamin as marker substances. The molecular weight of the NS,: preparation was thus found to be around 1300.
Thin-layer chromatography was ptrformed to compare the purified NS witl? known standard substances. 5-Hydroxytryptamine, 5-hydroxytryptophan, tryptophan, tryptamine, epinephrinc, norepinephine, tyramine, 5-hydroxytyramine (5 pg each) and NS, (in amounts showing an absorbance of 0.008 at 265 nm). were chromatographed as described in Materials and IIethods. Measured RI values
pH 4.3
pH 9.12 FIG. 8. Acrplamide
gel electrophoresis
of purified
NS at acid
and alkaline
pH levels.
CEPHALOPOD
CARDIOEXCITOR
275
NEUROHORMONE
FIG. 9. Spectrum of purified NH. Curve 1, NE& dissolved in distilled water; curve 2, N& in acidic (HCl) solution, pH 1; curve 3, NS6 in alkaline (NaOH) solution, pH 11. Spectra were recorded in l-cm lightpath opt.ical cells using an Optira recording spectrophotometer, Model CF4R.
after detection with the modified Ehrlich Freagent appear in Table 3. NS cannot be identified with any of these standards. It shows a dark blue fluorescence under uv light and a blue color (becoming brown in the next 2 hr, at room temperature) when colored with the modified Ehrlich reagent.
trast with the maximum at 549 nm found in the control experiment with N-acetylneuraminic acid as standards, an absorption maximum at 532 nm, which, according to Warren, is characteristic for 2-deoxyribose. DISCUSSION
_Pa,rtial Further Characterization Indole groups appear to be present in NS, as revealed on chromatograms by the Ehrlich or the modified Ehrlich reagents. The reaction for amino sugars was only slightly positive. NS, preparation, subjected to the thiobarbituric acid method of Warren ( 1959)) specific for sialic acid, gave, in conTABLE THIN-LAYER
NS
3
CHROMATOGRAPHY OF P~RIFIKII AND VARIOUS SUHSTANCW
Substance
RI
N& Epinephrine Norepinephrine o-Tryptophan 5-OH-oktryptophatl 5-OH-Tryptamine Tryptamine 5-OH-Tyramine Tyramine a For experimental
___ details, see text.
0.32 0.46 0.48 0.54 0.44 0.60 0.67 0.5ti 0.63
Extracts of the innervated region of the vena cava show a conspicuous stimulating action on the isolated heart of all the cephalopods studied, suggesting that the NVS system has the function of elaborating a neurohormone, NS, involved in the regulation of cardiac activity. Many observations support this suggestion. (1) The NSV system in cephalopods has an anatomical position and structure appropriate for releasing substances into the blood: it possessesa dense network of nerve fibers with a high concentration of elementary neurosecretory granules in close proximity to the blood stream. In other words, the NSV system is, anatomically, a neurohemal organ. (2) Extracts of the neurohemal organ have cardioexcitor effect. (3) The positive inotropic and chronotropic effect is longlasting, which is a characteristic attribute of endocrine factors and neurohormones. In addition to the long-lasting cardioexcitatory factor, a transient cardioexcitatory
276
RLANCH!,
NOVIELLO,
effect was found by Berry and Cottrell (1970) using extracts of different blood vessels of Eledone. They suggested, therefore, the presence of two cardioexcitatory factors. Extracts of other vessels, in the present work, were devoid of any activity, presumably because our extraction procedure (see Materials and Methods) allows a more selective extraction of the content of the nerve terminals, breaking down the axon membranes. (4~ NS is effective at very low concentrations and maintains its activity after several purification rtel)s. At, high concentrations it does not damage the perfused heart stopping it, revcrsihlp, in systole. (5) Electrical stimulation of t,hc NSV nerves liberates a subatancc qualitatively similar to that present. in t’hc cxtracts, suggesting that the acti\-(1 I)rinciplt contained in the nerve endings ran bc rcleased into the blood by nerv(l impulses. ~6) In all cephalopod!: examined a cardioexcitatory suhstancc was found in the extracts of the vena cava. Thtl (axtract’s were active on the heart of the same and of different cephalopod species. The active substance extracted from the NSV system of octopods appears to bc much more concentrated or potent than that, estractcd from decapods. This observation may have an explanation at the anatomical level: as described by Alexandrowicz (1965) nerve endings are denser in Octop,us and Eledone than in &pin. Thus ortopod extracts of NSV may contain more NS. On tht other hand, the decapod heart is more Fensitivr than that of ortopods. There is some evidence that the cardioexcitatory neurohormone is released in response to stress, as suggested hy Berry and Cottrell (1970) for Eledone cirrosa. Moreover, Martin (1968) observed that the ncurosecretory granules hccame electron transparent in an Octopus fixed after stress. Another experiment supporting this hypothesis is that of Blanchi (196910): blood taken from the vena cava or from any vessel of the animal caught from its tank in usual manner and sacrificed, had a cardioexcitatory action qualitatively similar to that of NS, when tested on an isolated, perfused heart; by contrast, blood
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taken from tentacles cut off very quickly did not affect the beat. This observation could indicate that release of NS into the blood results from the stress experienced by the animal when it is captured and killed. The observations together make it po+ sible to conclude that the NSV system has a neurosccrctory function as suggested h); Alexandrowicz (1965) and Martin (1968 I. Since NS can be easily released into the blood and rapidly carried through the whole body, cardiac regulation may be onl;\ one of it* functions; the role of NS may bc comparable to t,hat of epinephrine in vertebrates, influencing a number of physiologr ical activities in thrh animal. Such other functions remain to be investigated. The specific activity of the active principle has prcscntly hem increased about. 95-fold. Tbc substance shows a rather low molecular weight and cannot be identifier! with any of the well-known active euhstances found in several tissues of cephalopods. With respect to the problem of the structure of NS, it is of interest that the suhstance resists heating, but that the biological activity is almost completely destroyed hy digestion of crude extract with Pronaer. This may indicate that at least some pel)-’ tide bonds are present, in the molecule of the act.ive principle. The presence of indole groups (tryptophan?), as revealed by the positive Ehrlich reaction, is in line with the hypothesis of a partially peptidic nature of ?JS. On the other hand, despite’ t,hc only slightly positive reaction fol amino sugars, some carbohydrate moiety seemsto he present also, since the NS wastained with Astral blue and the reaction for carbohydrates was positive within thcs active fractions elutcd from Bio-Gel P-2. Further work is necessary to elucidatt the chemical nature of the active substancr isolated from the NSV system of Ocfop~~~~~ vulgaris. ACKNOWLEDGMENT We are very a critical reading suggestions.
grateful of the
to ProfessorFlorer fol manuscript
and
for
kind
CEPHALOPOD
CARDIOEXC !ITOR NEUROIIORMONE
REFERENCES J. S. (1964). The neurosecretory system of the vena cava in Cephalopoda. I. Eledone cirrosa. J. Mar. Biol. Ass. U. K. 44,
%EXANDROWICZ,
111-132. ALEXANDIKIWICZ,
J. S. (1965). The neurosecretory system of the vena cava in Cephalopoda. II. Sepia oficinalis and Octopus vulgaris. J. Mar.
Biol. Ass. U. K. 45, 209-228. ANDREWS, P. (1965). The gel-filtration
behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 59%606. BAILEY, J. L. (1962). “Techniques in Protein Chemistry,” pp. 295-299. Elsevier, Amsterdam. I~UXARI, N., AND MAZZI, V. (1966). “Manuale di Tccnica Microsropica,” 6th rd.. p. 247 Societit E:ditrice Libraria. Como. HERRY, C. F., AND COTTBELL, G. A. (1970). Neurosecretion in the vena cava of the Cephalopod Eledone cirrosa. Z. Zelljorsch. Mikrosk. Anat. 104, 107-115. BLANCHI, D.
(1969a). Esperimenti sulla funeione de1 sistema neurosecretorio della vena cava nei Cefalopodi. Nota I. Boll. Sot. Ital. Biol.
Yper. 45, 1615-1617. I%L.~NCHI, D. (1969b).
Esperimenti sulla funzione de1 sisteme neurosecretorio della vena cava nei Cefalopodi. Nota II. Boll. Sot. Ital. Bbl. Sper. 45, 1617-1619.
277
L. A.. AND MORGAN, W. T. J. (1933). A calorimetric method for determination of glucosamine and chondrosamine. Biochem. J. 27,
ELSON,
1824-1828. HODGE, J.
E.. AND HOFREITER, H. T. (1962). “Methods in Carbohydrate Chemistry” (R. L. Whistler and W. L. Wolfrom, eds.), Vol. 1. p. 380. Academic Press, New York. MARTIN, R. (1968). Fine structure of the neurosecretory system of the vena carn in Octopus Brnin MOORE,
Res.
8, 201-205.
S., AND STEIN, V. H. (1948). Photometric ninhpdrin method for use in the chromatography of amino acids. .1. Biol. Chem. 176, 367368. ORNSTEIN, L. (1961). Disc electrophoresis. Canal Industrial Corp., Bethesda, Maryland. REISFELD, R. A., LEWIS, V. J., AND WILLIAMS, D. E. (1962). Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature (London) 195, 281-283. SMITH, I. (1969). Chromatographic and electrophoretic techniques. Vol. I, pp. 253-254. Heinemann, Medical Books, London. WARBURC, O., AND CHRISTIAN, W. (1942). Isolierung und Kristallisation des GLrungsferment Enolase. Biochem. Z. 310, 384471. WARREN, L. (1959). The thiobarbituric acid assa? of sialic acid. J. Biol. Chem. 234, 1971-1975.
AND
COMPARATIVE
ENDOCRINOLOGY
A Neurohormone
21, 267-277 (1973)
of Cephalopods DONATELLA AND
Institute
with
Cardioexcitatory
Activity
BLANCHI, LUIGI NOVIELLO, MASSIMO LIBONATI’
of General Zoological
Physiology, University Station. Naples, Italy
Received September
of Turirc,
22. 1972
A neurohormone (NS) with long-lasting cardioexcitatory action on the isolated heart of cephalopods, has been extracted from the neurosecretory system of the vena cava (NSV system) of several cephalopod species. Electrical stimulation of the NSV nerves to the vena rava liberated a cardioexcitatory substance. Cardioexcitor extracts from Octopus vulgaris have had their activity increased 95 times by purification procedures. The resulting material is homogeneous on electrophoretic analysis, shows an absorbance maximum near 265 nm, and has a molecular weight of about 1366. NS is not identifiable with any ratecholamine or indole compound lmown to be present in cephalopod tissues.
The system of nerves which reach the anterior part of the vena cava of cephalopods, ending as a dense network of nerve fibers in close proximity with the blood stream, was first described by Alexandrowicz (1964, 1965) and named by him the neurosecretory system of the vena cava (NSV system). Axons of this system originate from nerve cells located in a layer of the palliovisceral lobe of the brain and in paired ganglionic trunks (lateral and medial NSV trunks) which emerge from the visceral lobe with the posterior infundibular and the visceral nerves, respectively. NSV nerves from these trunks 1:enetratc the muscular wall of the vena cava, forming a fine neuropile adjacent to the inner surface of the vessel. Martin (1968)) examining the fine structure of the NSV system in Octopus, demonstrated the presence of electrondense granules similar to those described in known neurosecretory tissue. Both authors suggested a neurosecretory function for the system. Blanchi (1969a,b) investigated the postu’ Present address: Laboratory of Biological Chemistry, Faculty of Sciences, Naples. 267 Copyright @ 1973 by Academic Prr::s. Inc. .4ll rights of reproduction in any form reserved.
lated secretory function and the physiological role of the NSV system with hearts isolated from various cephalopods. Extracts prepared from the innervated region of the vena cava, when added to the perfusion fluid, increased the frequency and amplitude of cardiac beat. It was suggested that the NSV system has the function of liberating a neurohormone involved in the regulntion of cardiac activity. Similar results and conclusions were presented by Berry and Cottrell (1970) after their study of fine structure of the neuropile of the vena cava of Eledone cirrosa. This paper describes further efforts to characterize and identify the active material of this neurosecretory system. In the first part a more detailed examination of function of the postulated neurohormone is described, in the second part a description of its gross chemical properties is presented. MATERIALS
AND
METHODS
Preparation of eztructs. Extracts were from the anterior innervated portion of cava of Octopus vulgaris Lam., Octopus Risso, Bledone moschata Lam., Sepia
prepared the vena mucropus officinalis
268
BLANCHI,
NOVIELLO,
L., and Loligo vulgaris Lam. as follows: a single vein was dissected from the experimental animal, cut longitudinally and immersed in 5 ml of distilled water for 15 min at room temperature (crude extract). Extracts of the abdominal aorta, prepared under identical conditions, were used as controls. The crude extracts were immediately used or stored at -30°C until use, berause at room temperature they lost activity. Bioassuy. Isolated and perfused hearts from the five species mentioned above were used for bio!ogicnl assays. Routine assays of activity of ihe neurosccretory substance, especially during the purification procedure, employed hearts from Octopus vnlgmis. The isolatrtl hearts were cannulated through the abdominal aorta and attached by the efferent branchial rcssels to a photoelectrical transduccxr directBy connected to the dc amplifier of a physiograph (Four A, E.M. Instruments Co., Inc.) ; heart recordings were isotonic. The hearts were perfused at a rate of about 20 ml/min. at constant pressure, with oxygenated sea water. Usually lo-50 ~1 of extract or of any fraction from the purification steps were diluted to 1 ml with filtered sea water and were injected into t,he perfusion system, not far from the heart: the dead time of the perfusion system was about 7 sec. In this way the injected solutions were diluted before reaching the heart and were washed out quickly thereafter. It was, therefore, not possible to determine absolute threshold. Concentrations givm for any testrd substance refer to the injected solution (1 ml) and are given in terms of units, 1 unit corresponding to the cardioexcitatory action of 10 ng of 5-hydroxytryptamine (5-HT). Spectrophotometric procedures. Absorbance at 265 nm (determined with a Zeiss PM& 11 spectrophotometer) was used to follow the active principle during the purification procc,dure or to its concrniration in test indicate, nlt,crnatively, solutions. The wavelength of 265 nm corresponds to the maximal nbsorhancr (see Fig. 9) of purified fraction of NS, as determined in pilot experiments. Chromatography. Unless otherwise specified. all ol)erations wrre carrirrl out at 0-4°C. Gel chromntograpl~y was prrformcd using Srph:&r G-75 superfine (Pharmacin Upl’snla) , pnckt~l in a K 15/90 c~olumn. 78 cm high, cctuilihrnted with 0.2 M NH,HCO,,, pH 7.S. Chromatography through Bio-Gel P-2 (Bio-Rad) was performed using bidistilled water as eluent. A flow rate of 10 ml/hr was obtained with a constant-volume pump (LKB-10200 Perspcx peristaltic pump) ; 2.5 ml fractions were collected. The Fo of the column was always determined using blue dex-
AND
LIBONATI
tran. Chromatography on CM-Sephadex C-25 (Pharmacia Uppsala) was conducted on columns equilibrated with 0.01 M Tris.HCl buffer (buffer A) at pH 8 (conductivity of 6-8 millimhos/cm). il stepwise grsdient between 0.1 and 1.0 M NaCl was used for elution (Fig. 7). Preparative chromatography was performed on thin-layer plates of Silica Gel F,,, (Merck, 20 X 20 cm) with a thickness of 0.25 mm. Development was performed one-dimensionally with nhutanol, acetic acid, and water (120:30:50) for 4 hours at, room temperature. Spots were detected by spraying with a solution of 0.1% Ce(SO,), in concentrated HSO, or with Ehrlich reagent (sometimes modified according to Smith, 1969). followed b,v heating at, 110°C for 4 min. Gel-electrophoresis. (A) Cellogel strips, 2.5 X 17 cm, were buffered in sodium Verona1 (8.24 g/liter) at pH 9.6 for about 2-3 hr (excess of buffer being rrmoved by gentle pressure with a filter paper sheet), and electrophorcsis was carried out at 200V for 75 min. Strips were then stained in an Astral blue solution (1% in 1% acetic acid; Beccari and Mazzi. 1966) for 12 hr and destained with SC% acetic acid. (B) Cylindrical polyacrylamide gels (Ornstein. 1961), 5% w/v, 6.5 mm in diameter, were used. Samples of the most purified fraction (NS,, Table 1. pool of several preparations), 50 or 100 ~1 in volume. corresponding to 0.05 or 0.1 OD units at 265 nm, rrsprclivrly, were mixed with equal volumes of 5O’i (w,‘v) glycerol and layered on the top of grls. Electrophoresis was carried out with a constant current of 5 V/cm per tube, at room t,rmperature. for about. 60 min. The buffers used were 0.05 Jf tris.glycine, pH 9.2, or palanine buffer of Reisfeld et al. (1962). Gels were stained with Ast,ral blue for 12 hr and destained in 5% acetic acid for 24 hr. Other nr,e&~Z.s. The following analytical methods were used: for carbohydrates the method of Hodge and Hofreiter (1962); for amino nitrogen that of Moore and Stein (1948). Proteins were determined by ihe 280:260 nm absorption quotient (Warhurg and Christian, 1942) or by the microbiurct method (Bailey, 1962). The method of Warren for sialic acid (1959), and that of Elson and Morgan (1933) for hexosamines were usrd. The ionic pattern of the CM-Sephadex C-25 chromatography was determined with a conductivity meter, t,ype CDM2 (Radiometrr. Copenhagen). Chemicals. Ninhydrin and acetylacetone were from Merck. Other chemicals from Sigma Chem. Co. Pronasc ~vas purrlrnsed from Koch-Light Lab. Ltd.: Astral blur from Gurr’s Ltd., London. England.
CEPHALOPOD
CARDIOEXCITOR
NEUROHORMONE
t NS
269
t
I 1 min
t 5-HT FIG. 1. Isolated heart of Octopus vu[garis; arrows indicate NS (absorbance at. 265 nm, 0.436) and 5-hydroxytryptamine,
RESULTS
Biological Characterization of the Vena Cava Extracts of Octopus vulgaris The remarkable cardioexcitatory action of the extracts of the vena cava-innervated region consists of a long-lasting positive inotropic and chronotropic effect. The rtsponse grows to a maximlim 30-45 set after the injection and then persists for as long as 20 min (Fig. 1). With higher concentrations of extracts an increase in tone can be observed, which finally leads to systolic
t 10
'
125
Fro. 2. Action of heated NS and indicate application of test solutions. of NS corresponds to an absorbance Records were cut and rearranged 2030 min. Note that the time from to first. noticeable response is longer
'
150
application of tesl. solubons: 500 ng. Vertical calibration
heated : 10 g.
(see text)
arrest. After washing, the heart starts beating again. Repeated introduction of the extract results in a progressive reduction of the response. Control experiments were carried out by injecting aorta extracts; in this case no variation of the beating was observed. The action of Octopus vulgaris NS was compared with that of epinephrinr, norepinephine, and 5-hydroxytryptamine : these substances have a marked positive inotropic and chronotropic action on the heart of O&opus, but the effect is transient,
I
I100
'
1200
of 5-hydroxytryptamine (5-HT) on the isolat.ed Oclopus heart. Arrows Indicated NS amounts are expressed in microliters of extract: 1 ~1 of 1.09 X lOa3 at 265 nm. 5-HT amounts are given in nanograms. to facilitate dose-response comparisons: at X, record was stopped foi injection to maximum response is approximately constant, though time for more dilute injections.
“70
t!I,.\h-CHI,
NOVIELLO,
AND
LIBONATI
t cw 1 min VW. 3. Action heart of 0ckq1u.s
of 50 ~1 of “uJllt,rol water’J (CR) and “stimulat~iori At S, record was stopped for 20-30 min.
lasting not more than 2 min. The acccleration obtained with 5-hydroxytryptnmine was often followed, during washing, by a period of depressed activity. In Fig. 2 the action of NS is compared to that of 5-HT. Electrical
water”
(hw)
isee lest)
on the isolaled
uulgaris.
Xtimulation
Expehnlents
The vena cava, the medial ncurosecretory nerves (NW nerves) with the visceral nerves were carefully dissected out and put in 5 ml of oxygenated sea water; SSV nerves were picked up on silver electrodes and stimulated with trains of pulses every 10 see, 1 WC duration, at a frequency of 100 Hz; each pulse with a duration of 0.5 millisec and an amplitude of 2-5 V. After stimulation for 15, 30, and GOmin the bathing medium (“stimulation wakr”) was assayed on the heart. (‘ontrols con>istccl of
identical preparations without stimulation (L’control water!‘) Figure 3 shows the effect of successive injections of 50 ~1 (in 1 ml of sea water) of “control water” and of “stimulation water,” from a preparation that, had been stimulated for 30 min as dcscribed abovca. An increase of nmplituclc and frequency follows I)oth injections, but a significantly stronger effect can be observed in th(, rnsc of the “stimulation water.” With smaller closes or shorter periods of stimulation and immersion (of the vena envat “control water” has no cffeet, while “stimulation water” ohtaincd under iclent8ical conditions, has a cartlioexcitatory action qualitatively similar to that produrcd by the vrna cava extracts. The small exrit,atory effect of the ‘Lront’rol water” indicatcb:: that some NS i:: rclcasrd by the tissue n-hen uiistimulntcrl.
1 min
FIG. 4. (A) A&m of cr~tde NS of Octopus vu1gari.s (0.) and of Elrdonc moschnta (E;.) (absorbance at’ 265 nm, 0.350) on the isolated heart of Eledone. (B) Action of crude NS of Sepkx (S.) and of Octopus ~lgaris heart of Scpiu. Arrows indicat,e application of test solu(0.) (absorbance at 265 nm, 0.020) on the isolated Gons. At X, record wa.s stopped for 20-30 min. The same Octopus extract, was injected in both hearts, hlit it, was dilut,ed 17.5 times to be tested on Sepia heart.
CEPHALOPOD
Procedure >Crude ext,rtlction Itea+ Sephades G-75 Bio-Gel P-2 CRI-Rephades C-25 Thin layer (preparative)
Frac(ioll NS .;I KS, NP, h’si, ?u’S&
CARDIOEXCITOR
Volume (ml) 110.0 10s.0 4 2 1 0.
Total Imits
Yields c”; 1
723 43XS 1 SO4 740 260 1.31
100. II0 B” so 21 !I0 10 “:I :: .60 2.10
:3 0 3 1
“71
NEUROHORMOSE
Total :hsorl~ance nt 26.5 11111 550 0 Y4S.4 “8.6 1.4 0.4 0.12
Specific activity units ’ absorbance at, 265 nm 1:i. 1 1x2 76.4 525.6 650.0 1250.0
Relative pllrification
1.4 5,s 40. :I 49.6 95 .4
-
Assays of Vena Cazla Extracts on lIonor Species or on Different Species of Cephalopods
Incubation carried out with trypein or papain under the same conditions did not affect the activity of NS on the isolated Octopus heart.
Extracts were prcpartd from the venae cavae of the 5 species easily available in Purification of the Vena Cava Extract &he Bay of Naples (Octopus vulgaris, Octopus ~w~c~opus, Eledone moschata, Sepia A typical purification procedure is sumoficinalis, and Loligo vulgaris). The action marized in Table 1. Protein concentration of NS of each species was tested on the I 1 heart of the same and on that of the other -ABSORBANCE 2E5nm species: increase of frequency and ampli~.-.~ ABSORBANCE 2BiJnm tude of the beat was always observed. Figure 4 shows that the injection of crude .,extract produced a marked effect in the case of the direct and of the crossed experiments. The comparison was made on the basis of total activity per vena cava and mtasuring the absorbance at 265 mn of the injected extracts. The NS of any octopod sllecies is equally cffectivc on the hearts of other octopods, while it must bc diluted lo-20 times for similar effects on the tlecapods Sepia and Loligo. In like manner, 301 NS from the two decapod species are equally effective on the decapod hearts but must be concentrated at least ten times *to produce marked effect on octopod hearts. nigestim of Cmde Esfrncf u\ifh Pronase Incubation of 0.5 ml of crude extract with 0.2 mg of Pronase at 37°C for 2--3 hr destroyed 8&90% of the activity present ‘in the untreated extract. Control expcriments with equivalent amounts of pronase solutions in 1 ml of sea water had no effect on heart heat.
FIG. 5. Gel-chromatography through Sephades G-75. NS? preparation (absorbance at 265 nm, 126) was applied onto a 1.5 X 78 cm column and eluted at 4°C with 0.2 III NHIHCO~, at pH 7.8. Flow rate, 10 ml/hr. Active fractions (62-77) were pooled.
272
BLANCHI,
NOVIELLO,
has been taken as an index of active material concentration through the first purification steps. The “specific activity” was defined as the ratio between biological activity unit and the absorbance at 265 nm, where the active substance (partially purified in pilot experiments) showed it’s maximum. Examples presented in the figures have
46
-
36
-
26
-
16
-
6.0
AND
LIBONATI
been taken from different, individual experiments. Xtep 1. Crude extract (NS,) was prepared by the procedure described under Materials and Methods (Preparation of extracts). Step Z?. The crude extract was heated in a boiling water-bath for 15 min, and the insoluble material was removed by cen-
a
s2.0 r u ml.8 a
-
18
El.6
-
16
u 1.4
-
1.2
-
1.0
-
0.8
-
0.6
-
8
I
6
I,Y :’J: -i
40
50
60
70
80
(
FRACTIONS FIG. 6. Chr0matograph.v of N& preparation (absorbance at 265 nm, 260) through a Bio-Gel P-2 column (1.5 x 78 cm). Flow rate, 10 ml/hr; temperature, 4°C; 2.5-ml fractions were collected. Shaded area indicates fractions containing biological activity (activity units/ml X 10). Fract.ions IS-26 were pooled (NW -- m, Absorbance at 265 nm; - - -, absorbance at’ 280 nm; O-0, carbohydrat’es (&ml); . . ., amino nit,rogerl (~moles/ml).
CEPHALOPOD
CARDIOEXCITOR
tivity ; the third peak represented the active fraction (NS,). A typical experiment is shown in Fig. 5. Step 4. Fraction NS, was now lyophilized, pooled wit,h others prepared from similar experiments, dissolved in bidistilled water, and applied onto a Bio-Gel P-2 column. At least 4 peaks (absorbance at 265 nm) emerged (Fig. 6). Activity was found only in the first peak. The material was lyophilized again and dissolved in bidietihed water (XS,). Step 5. Fraction NS, was applied to a
trifugation at 27,000g for 15 min at M”C. ‘Supernatant solutions were lyophilized !NSA. The activity is unaffected by heating, a sample of heated extract being equally active as a same sample of crude extract. Step S. Preparation NS, was dissolved in >bidistilled water and applied onto a Sephadex G-75 ‘Superfine” column. The effluent showed three peaks, absorbing at 280-260 nm ; t,wo of them, corresponding to about 90% of the total protein input., were exeluded since they were devoid of any acbufferA
bufferA+ 0.1 M NaCl
50ml
50ml
273
NEUROHORMONE
buffer A + 0.5 M Na C I
buffer A+ 1.0M Na Cl
100ml
1.4 -
I 55
1.3 .*I’
I:.
...:’,.*.4.. . .. . ._.. . . . . . .
1.2 -
1
1.0 -
40
:..“.
. . . ..a...
. . . . .
z-
..-.
0.9 -
,+; :: ::: .
z 0.6 z =: 0.7 cp u
50
45
, ,
1.1 -
w v
-1
100ml
:
; :
-35
,c a
-30.
& m
-25
z
::
i
:: :.i
:
;
0.6 0.5 0.4 0.3 0.2 0.1
25
50
75
100
125
150
175
200
225
250
275
ml of eluate FIG. 7. Chromatography of NS, preparation (absorbance at 265 nm, 18.4) through a CM-Sephadex C-25 column (1 X 10 em). Elution was carried out by stepwise gradient using sodium chloride in 0.01 M trisHC1 buffer (buffer A), pH 8; 2.5-ml fractions were collected. Flow rate, 10 ml/hr; temperature, 4°C. Shaded area indicates active fractions (between 2.5 and 25 ml of eluent) (activity units/ml X 10). --, Absorbance at:265 nm: - - -, absorbance at. 280 nm; . . . ., conductivit)y (mmhozcm).
274
BLANCHI,
NOVIELLO,
CM-Sephadex C-25 column and eluted with a stepwise gradient (Fig. 7). Activity was found in the first 30 ml of eluate. The active fractions were pooled and lyophilized (NS,) . Step 6. Preparative chromatography on thin-layer plates. Fraction NS, was applied on a Silica Gel F,,, plate (see Materials and Methods). Two bands were detected; only one of them, with a lower Rf, was active (NS,) . Criteria
AND
LIBONATI
TABLE
2
DETERMINATION OF THE MOLECULAR WEIGHT OF PUKIFIED NS ACCORDING TO ANDREWS (1965)= Substances Riboflavin Coenxyme A NS Cyanocobalamin
Elution volume
Molecular weight
120.7 68.0 47.6 42.5
376.4 767.6 1280.0 1355.4
(found
)
a Bio-Gel P-6 column, 0.9 X 90 cm, equilibrated and eluted with distilled water. Flow rate, 10 ml/hr. Fractions of 1.7 ml were collected. The column was subsequently loaded with 0.5 mg of riboflavin, 0.2 m,$ of CoA or cyanocoba.lamin and with N& (pool of several preparations; t,otal absorbance at 265 nm = 1.2). The elution pat,tern of the various substances wm det,ermined by absorbance at. the following wavelengths: 450 nm for riboflavin, 260 nm fat coenzyme A, 360 nm for cyanocobalamin, 265 nm for N&.
of Pwity
The electrophoresis pattern of the NS, preparation on Cellogel shows a single band, stained with Astral blue, even if rather large amounts of the substance arc applied. Analytical disc electrophoresis of NS,, also shows a single band (Fig. 8). Electrophoresis was carried ol7t ad described under Materials and Xfethods. Each gel was split longitlldinally: one half was Ultraviolet Absolptioti Spectrum stained with Astral blue, the other was The uv spectra of NS, dissolved in water sliced in 2-mm pieces. Each piece was then or at pH 1 show a broad maximum around homogenized in 1 ml of bidistilled water. 265 nm (Fig. 9, curves 1 and 2). At pH 11 After centrifugation at 10,OOOgfor 10 min. this maximum disappears, but a shoulder t,he supernatant was tested for biological activity under standard conditions. The appears in the region between 240 and 255 cardioexcitatory activity was found in the nm (Fig. 9, curve 3). The absorbance ratid region of the gel corresponding to t.he 280:260 nm, as measured from the spertrum, was 0.84. stained band of the counterpart. Behavior
Molecular Weight
Chromatographic
The molecular weight of the purified NS was determined by the gel-filtration m&hod described by Andrews (1965). A Bio-Gel column (see Table 2 for experimental details) was loaded with NS, and SUCCCSsively, under identical experimental conditions, with riboflavin, coenzyme A, and ryanocobalamin as marker substances. The molecular weight of the NS,: preparation was thus found to be around 1300.
Thin-layer chromatography was ptrformed to compare the purified NS witl? known standard substances. 5-Hydroxytryptamine, 5-hydroxytryptophan, tryptophan, tryptamine, epinephrinc, norepinephine, tyramine, 5-hydroxytyramine (5 pg each) and NS, (in amounts showing an absorbance of 0.008 at 265 nm). were chromatographed as described in Materials and IIethods. Measured RI values
pH 4.3
pH 9.12 FIG. 8. Acrplamide
gel electrophoresis
of purified
NS at acid
and alkaline
pH levels.
CEPHALOPOD
CARDIOEXCITOR
275
NEUROHORMONE
FIG. 9. Spectrum of purified NH. Curve 1, NE& dissolved in distilled water; curve 2, N& in acidic (HCl) solution, pH 1; curve 3, NS6 in alkaline (NaOH) solution, pH 11. Spectra were recorded in l-cm lightpath opt.ical cells using an Optira recording spectrophotometer, Model CF4R.
after detection with the modified Ehrlich Freagent appear in Table 3. NS cannot be identified with any of these standards. It shows a dark blue fluorescence under uv light and a blue color (becoming brown in the next 2 hr, at room temperature) when colored with the modified Ehrlich reagent.
trast with the maximum at 549 nm found in the control experiment with N-acetylneuraminic acid as standards, an absorption maximum at 532 nm, which, according to Warren, is characteristic for 2-deoxyribose. DISCUSSION
_Pa,rtial Further Characterization Indole groups appear to be present in NS, as revealed on chromatograms by the Ehrlich or the modified Ehrlich reagents. The reaction for amino sugars was only slightly positive. NS, preparation, subjected to the thiobarbituric acid method of Warren ( 1959)) specific for sialic acid, gave, in conTABLE THIN-LAYER
NS
3
CHROMATOGRAPHY OF P~RIFIKII AND VARIOUS SUHSTANCW
Substance
RI
N& Epinephrine Norepinephrine o-Tryptophan 5-OH-oktryptophatl 5-OH-Tryptamine Tryptamine 5-OH-Tyramine Tyramine a For experimental
___ details, see text.
0.32 0.46 0.48 0.54 0.44 0.60 0.67 0.5ti 0.63
Extracts of the innervated region of the vena cava show a conspicuous stimulating action on the isolated heart of all the cephalopods studied, suggesting that the NVS system has the function of elaborating a neurohormone, NS, involved in the regulation of cardiac activity. Many observations support this suggestion. (1) The NSV system in cephalopods has an anatomical position and structure appropriate for releasing substances into the blood: it possessesa dense network of nerve fibers with a high concentration of elementary neurosecretory granules in close proximity to the blood stream. In other words, the NSV system is, anatomically, a neurohemal organ. (2) Extracts of the neurohemal organ have cardioexcitor effect. (3) The positive inotropic and chronotropic effect is longlasting, which is a characteristic attribute of endocrine factors and neurohormones. In addition to the long-lasting cardioexcitatory factor, a transient cardioexcitatory
276
RLANCH!,
NOVIELLO,
effect was found by Berry and Cottrell (1970) using extracts of different blood vessels of Eledone. They suggested, therefore, the presence of two cardioexcitatory factors. Extracts of other vessels, in the present work, were devoid of any activity, presumably because our extraction procedure (see Materials and Methods) allows a more selective extraction of the content of the nerve terminals, breaking down the axon membranes. (4~ NS is effective at very low concentrations and maintains its activity after several purification rtel)s. At, high concentrations it does not damage the perfused heart stopping it, revcrsihlp, in systole. (5) Electrical stimulation of t,hc NSV nerves liberates a subatancc qualitatively similar to that present. in t’hc cxtracts, suggesting that the acti\-(1 I)rinciplt contained in the nerve endings ran bc rcleased into the blood by nerv(l impulses. ~6) In all cephalopod!: examined a cardioexcitatory suhstancc was found in the extracts of the vena cava. Thtl (axtract’s were active on the heart of the same and of different cephalopod species. The active substance extracted from the NSV system of octopods appears to bc much more concentrated or potent than that, estractcd from decapods. This observation may have an explanation at the anatomical level: as described by Alexandrowicz (1965) nerve endings are denser in Octop,us and Eledone than in &pin. Thus ortopod extracts of NSV may contain more NS. On tht other hand, the decapod heart is more Fensitivr than that of ortopods. There is some evidence that the cardioexcitatory neurohormone is released in response to stress, as suggested hy Berry and Cottrell (1970) for Eledone cirrosa. Moreover, Martin (1968) observed that the ncurosecretory granules hccame electron transparent in an Octopus fixed after stress. Another experiment supporting this hypothesis is that of Blanchi (196910): blood taken from the vena cava or from any vessel of the animal caught from its tank in usual manner and sacrificed, had a cardioexcitatory action qualitatively similar to that of NS, when tested on an isolated, perfused heart; by contrast, blood
AND
LIBONATI
taken from tentacles cut off very quickly did not affect the beat. This observation could indicate that release of NS into the blood results from the stress experienced by the animal when it is captured and killed. The observations together make it po+ sible to conclude that the NSV system has a neurosccrctory function as suggested h); Alexandrowicz (1965) and Martin (1968 I. Since NS can be easily released into the blood and rapidly carried through the whole body, cardiac regulation may be onl;\ one of it* functions; the role of NS may bc comparable to t,hat of epinephrine in vertebrates, influencing a number of physiologr ical activities in thrh animal. Such other functions remain to be investigated. The specific activity of the active principle has prcscntly hem increased about. 95-fold. Tbc substance shows a rather low molecular weight and cannot be identifier! with any of the well-known active euhstances found in several tissues of cephalopods. With respect to the problem of the structure of NS, it is of interest that the suhstance resists heating, but that the biological activity is almost completely destroyed hy digestion of crude extract with Pronaer. This may indicate that at least some pel)-’ tide bonds are present, in the molecule of the act.ive principle. The presence of indole groups (tryptophan?), as revealed by the positive Ehrlich reaction, is in line with the hypothesis of a partially peptidic nature of ?JS. On the other hand, despite’ t,hc only slightly positive reaction fol amino sugars, some carbohydrate moiety seemsto he present also, since the NS wastained with Astral blue and the reaction for carbohydrates was positive within thcs active fractions elutcd from Bio-Gel P-2. Further work is necessary to elucidatt the chemical nature of the active substancr isolated from the NSV system of Ocfop~~~~~ vulgaris. ACKNOWLEDGMENT We are very a critical reading suggestions.
grateful of the
to ProfessorFlorer fol manuscript
and
for
kind
CEPHALOPOD
CARDIOEXC !ITOR NEUROIIORMONE
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