J Comp Physiol B (1996) 165:518 526

9 Springer-Verlag 1996

D. J. Conklin 9 H. B. Lillywhite 9 K. R. Olson R. E. Ballard 9 A. R. Hargens

Blood vessel adaptation to gravity in a semi-arboreal snake

Accepted: 14 September 1995

Abstract The effects of vasoactive agonists on systemic

blood vessels were examined with respect to anatomical location and gravity acclimation in the semi-arboreal snake, Elaphe Obsoleta. Major blood vessels were reactive to putative neurotransmitters, hormones or local factors in vessel specific patterns. Catecholamines, adenosine triphosphate, histamine and high potassium (80mM) stimulated significantly greater tension per unit vessel mass in posterior than anterior arteries. Anterior vessels were significantly more sensitive to catecholamines than midbody and posterior vessels. Angiotensin II stimulated significantly greater tension in carotid artery than in midbody and posterior dorsal aorta. Arginine vasotocin strongly contracted the left and right aortic arches and anterior dorsal aorta. Veins were strongly contracted by catecholamines, high potassium and angiotensin II, but less so by adenosine triphosphate, arginine vasotocin and histamine. Precontracted vessels were relaxed by acetylcholine and sodium nitroprusside, but not by atrial natriuretic peptide or bradykinin. Chronic exposure of snakes to intermittent hypergravity stress (+1.5 Gz at tail) did not affect the majority of vessel responses. These data demonstrate that in vitro tension correlates with known patterns of sympathetic innervation and suggest

that catecholamines, as well as other agonists, are important in mediating vascular responses to gravitational stresses in snakes.
Key words Blood vessels 9 Gravity 9 Catecholamines 9 Vasoactive agents 9 Snake, Elaphe obsoleta Abbreviations ACH acetylcholine - ADA anterior

dorsal aorta" ANG II salmon asnl-valS-angiotensin II' A N P rat ileZ6-atrial natriuretic peptide 9 A TP adenosine triphosphate - A V T arginine vasotocin 9 B K human bradykinin 9 BL total body length 9 CA carotid artery 9 C O N T control ECso effective concentration producing 50% maximal response 9 EPI epinephrine 9 + Gz earth's gravity force 9 HI-G high gravity acclimation 9 HI K + 80 m M high potassium 9 J V jugular vein L A A left aortic arch - M D A midbody dorsal aorta M P V midbody portal vein- M S Mackenzie's solution" N E P I norepinephrine; pDz-log ECs0 9 PDA posterior dorsal aorta. P P V posterior portal veinR A A right aortic arch 9 S N P sodium nitroprusside

Introduction Changes in posture can impose dramatic hydrostatic challenges to hemodynamics in the peripheral vasculature of terrestrial vertebrates. By virtue of their long bodies and behavioral diversification, snakes provide a sensitive vertebrate animal for studies of cardiovascular response and adaptation to gravity (Lillywhite 1987). Valves appear to be largely absent from the larger veins of snakes, so both arterial and venous columns generate substantial hydrostatic gradients when vasculature is parallel to the gravitational force vector during vertical postures. In general, species of snakes having arboreal habits or a propensity for climbing (scansorial), when compared with aquatic or non-climbing species, demonstrate

D.J. Conklin ~ 9 H.B. Lillywhite2- K.R. Olson 3 R.E. Ballard 9A.R. Hargens (Ng) Life Sciences Division, NASA-Ames Research Center, Moffett Field, CA 94035-1000, USA
Present addresses:

Department of Biological Sciences, University of Notre Dame and South Bend Center for Medical Education, Indiana University School of Medicine, Notre Dame, Indiana 46556, USA z Department of Zoology, University of Florida, P.O. Box 118525, Gainesville, Florida 32611-8525, USA 3 South Bend Center for Medical Education, University of Notre Dame, Notre Dame, IN 46556, USA

D.J. Conklin et al.: Reactivity of snake blood vessels in vitro s u p e r i o r a b i l i t i e s to r e g u l a t e a r t e r i a l b l o o d p r e s s u r e , m a i n t a i n v i t a l o r g a n b l o o d flow, a n d resist e d e m a a n d b l o o d p o o l i n g in d e p e n d e n t v a s c u l a t u r e d u r i n g h e a d u p tilt ( S e y m o u r a n d L i l l y w h i t e 1976; L i l l y w h i t e a n d P o u g h 1983; L i l l y w h i t e 1985; L i l l y w h i t e a n d G a l l a g h e r 1985; L i l l y w h i t e 1993a, b). T h e a b i l i t y of s n a k e s to c o u n t e r a c t g r a v i t a t i o n a l d i s t u r b a n c e to the c i r c u l a t i o n d e p e n d s o n a suite of c h a r a c t e r i s t i c s , i n c l u d i n g physiological, morphological and behavioral adaptive t r a i t s [review: L i l l y w h i t e (1987)]. B o t h p h y s i o l o g i c a l p r o p e r t i e s o f b l o o d vessels a n d the m o r p h o l o g i c a l c h a r a c t e r i s t i c s o f v a s c u l a r a n d s u r r o u n d i n g tissues a r e i m p o r t a n t in c o u n t e r a c t i n g b l o o d p o o l i n g , w h i c h is t h e p r i n c i p a l p r o b l e m i m p o s e d b y p o s t u r e - r e l a t e d g r a v i t y force. Previous authors have demonstrated the presence of extensive adrenergic, cholinergic and peptidergic innerv a t i o n o f t h e h e a r t a n d v a s c u l a t u r e of s n a k e s ( D o n a l d a n d L i l l y w h i t e 1988; D o n a l d et al. 1990a, b; L i l l y w h i t e a n d D o n a l d 1994). I n t h e s e m i - a r b o r e a l y e l l o w r a t s n a k e (Elaphe obsolem), a d r e n e r g i c i n n e r v a t i o n inc r e a s e s in d e n s i t y a l o n g t h e s n a k e ' s l o n g i t u d i n a l axis a n d is m o s t d e n s e in t h e p o s t e r i o r a r t e r i e s a n d veins ( D o n a l d a n d L i l l y w h i t e 1988). T h i s i n n e r v a t i o n p a t t e r n i m p l i e s t h a t s t r o n g n e u r a l m e c h a n i s m s a r e i m p o r t a n t in m e d i a t i n g c i r c u l a t o r y r e s p o n s e s to h y d r o s t a t i c p e r t u r b a t i o n s t h a t o c c u r d u r i n g h e a d - u p p o s t u r e . T h e r e is, h o w e v e r , little i n f o r m a t i o n r e g a r d i n g t h e effects o f p u t a t i v e n e u r o t r a n s m i t t e r s , l o c a l f a c t o r s o r h o r m o n e s in various regions of the systemic vasculature. T h e p r e s e n t s t u d y w a s d e s i g n e d to d e t e r m i n e t h e r e a c t i v i t y o f s y s t e m i c b l o o d vessels to p u t a t i v e n e u r o transmitters, local factors and hormones. Isolated vasc u l a r r i n g s w e r e u s e d to e x a m i n e t h e r e s p o n s e o f vessels w i t h r e s p e c t to vessel type, l o c a t i o n a l o n g t h e l e n g t h o f the b o d y , a n d c h r o n i c e x p o s u r e to i n c r e a s e d g r a v i t y stress. Y e l l o w r a t s n a k e s , Elaphe obsoleta, w e r e u s e d in this i n v e s t i g a t i o n b e c a u s e o f t h e i r s c a n s o r i a l h a b i t s a n d k n o w n c a p a b i l i t i e s to r e g u l a t e h e m o d y n a m i c s w h e n s u b j e c t e d to a c u t e g r a v i t a t i o n a l stress i n d u c e d b y h e a d u p p o s t u r e ( L i l l y w h i t e a n d G a l l a g h e r 1985). E x p e r i m e n t a l a n i m a l s w e r e p e r i o d i c a l l y s u b j e c t e d to inc r e a s e d g r a v i t y stress b y i n t e r m i t t e n t c e n t r i f u g a t i o n w i t h t h e force v e c t o r in the h e a d - t o - t a i l d i r e c t i o n to d e t e r m i n e w h e t h e r a d j u s t m e n t s o f vessel r e a c t i v i t y o c c u r r e d in r e s p o n s e to i n c r e a s e d stress l o a d i n g . Acclimation to hypergravity

519

To evaluate vascular adjustment to gravitational stress, four snakes (454.2 + 81.8 g body mass; 141.5 + 13.1 cm BL; mean_+SD), which had been acclimated to intermittent hypergravic acceleration produced in the head-to-tail direction ( + Gz) on a 2.4 m diameter centrifuge, were used. During centrifugation, snakes were held within individually fitted acrylic tubes mounted parallel to the arms of the centrifuge and provided with ventilation ports and sufficient space for breathing as well as ca. 10 cm forward or backward movement. The tail end of a snake was positioned at a distance of 152 cm from the center of rotation. Confinement to the tube thus maintained the body in a straight position (except for limited sinusoidal curvature) with the head facing the axis of rotation. The force during centrifugation was measured with an accelerometer mounted on the centrifuge arm at a level even with the end of a snake's tail. Snakes experienced a gradient of Gz force equal to 0.02 Gz per cm of BL and the chosen maximum Gz force (1.5; measured at the tail end) was achieved following stepwise 3-rain increases of + 0.5 Gz. Snakes were exposed to the + 1.5-Gz centrifugal force for 15-20 min, three times a day, for 40 days, not including weekends. Four control snakes (331 _+ 54.4 g body mass; 128.7 + 10.7 cm BL) were placed in tubes and treated identically to the centrifuged snakes, except they were not subjected to increased G forces on the centrifuge. All snakes were kept in cages between centrifugations. At the end of the acclimation period snakes were killed with lethal intravascular injections of sodium pentobarbital (35-40 mg. kg-1 body mass; Abbott Chemical Co., Chicago, Illinois), and vessels were removed and placed in 4 ~ MS.

Isolated vascular rings Vessel segments (1-2 cm) from arteries (CA, ADA, MDA, PDA, LAA, RAA) and veins (JV, MPV, PPV) were removed from the same approximate locations in all snakes. CA and JV segments were removed from halfway between snout and heart (8.3 + 0.1% BL). ADA, LAA and RAA segments were taken within 1 cm of the confluence of the two arches (19.4 _+ 0.4% BL). MDA and MPV segments were removed halfway between the snout and the vent (40.1 _+ 0.8% BL). The PDA and PPV segments were removed halfway between the MDA segment and the vent (61.2 +_ 0.7% BL). The ventricle occupied a mean position of 16.7 + 0.3% BL which is typical of arboreal snakes [17.4 _+ 2.2% BL; Seymour (1987)]. Vessels were cut transaxially into 2- to 4-ram rings which were hung on 280!,tm diameter steel hooks in 20-ml smooth muscle chambers containing MS and bubbled with 95% 02: 5% CO2. Tension was measured with a Grass FTO3C force-displacement transducer and recorded on a Gould Series 8000S polygraph with sensitivity set to detect changes of 5 rag. Resting tension of 300 mg (veins) and 500 mg (arteries) was applied to the vessel rings for at 1east i h before experimentation. During this hour, rings were precontracted with EPI or NEPI (10 -6 tool. 1-~), washed three times, and baseline tension re-established for at least 30 min before further exposure (Olson and Meisheri 1989). Two rings from each vessel were contracted with cumulative doses of agonists (ANG II; AVT; EPI; NEPI) until maximal tension was achieved. A single ring was used for two dose response curves, one with a catecholamine and another with a peptide (ANG II or AVT). Rings were also exposed to a single dose of 80 retool- 1-1 HI K + and ATP (10- 5 mol" l - 1) or histamine (10- s tool. 1-1) and between exposures rings were washed three times with MS and tension readjusted to baseline over 1 h. Cumulative dose-response curves were performed on arteries and veins to determine ECso. The ECs0 was extrapolated from individual dose-response curves and converted to the pD2 ( - log ECso). Contractions were quantified as active tension (rag) per milligram of ring wet weight (Conklin and Olson 1994a).

Materials and methods

Animals Yellow rat snakes (Elaphe obsoleta quadrivittata) of mixed sexes were shipped by air from a supplier in Florida and maintained in the Life Sciences Building at NASA-Ames Research Center for several months prior to measurements. Snakes were kept in fiberglass cages with ad tibifum access to water and were fed mice weekly. Acclimation and experimentation air temperatures were maintained between 23 and 28 ~

520 Rings with stable contractions at the end of cumulative doses (maximal EPI or NEPI, 10 -4 mol.1-1) were exposed to a single dose of vasorelaxant drugs (ACH, 10-r A N G II, 10-7mo1"1-1; ANP, 1 0 - S t o o l - l - i ; ATP, 1 0 - S t o o l . l - l ; BK, 10 -7 m o l ' l 1; histamine, 10 -s m o l ' l - 1 ; or SNP, 10 .4 mol'l-1). The mechanism of ACH-induced relaxations was investigated in precontracted JV and MPV by incubation with indomethacin (10- 5 tool" 1-1) or methylene blue (10- s tool" 1-1) 15-30 min prior to addition of ACH. Relaxations are reported as percent reduction of active tension (Conklin and Olson 1994b). Experiments using rings from the same snake were conducted over 24-36 h.

D.J. Conklin et al.: Reactivity of snake blood vessels in vitro

Chemicals Composition of MS (in m m o l 1 1) was: 115.0 NaC1, 20.0 NaHCO3, 3.2 KC1, 1.3 CaC12, 1.4 MgSO4, 3.1 Na2HPO4, 16.7 glucose; pH 7.5. H I K + M S contained (in mmol-1-1) : 38.8NAC1 and 80.2KC1. ANP was a generous gift from Dr. E. Blaine (Monsanto). All other drugs were purchased from Sigma Chemical Company (St. Louis, Missouri).

Statistics Comparisons between vessel responses of C O N T and HI-G snakes were made with the Wilcoxon ranked-sum test with the significance level set at P <_ 0.05. When a statistical difference was not detected between C O N T and H I - G snake responses, the data were pooled for that vessel and agonist. Data from a group with too few snakes for comparison (n < 3 per group, C O N T or HI-G) were also pooled. Multiple comparisons between vessels or agonists were made with pairwise, one-way ANOVA of ranks. All values are expressed as mean _+ standard error (SE).

Results Tension development Active tension generation was agonist- and vessel-specific (Fig. la-g). In general, arteries responded to catecholamines in a pattern associated with their anatomical location (Fig. la, b). The most posterior artery tested, PDA, generated the greatest tension while the most anterior artery, CA, produced the least. Vessels between these two arterial segments produced intermediate levels of tension. Veins were also stimulated by catecholamines, and JV and M P V tension normalized for wet weight was similar in magnitude to that of some arteries. The M P V produced the least tension of all vessels but displayed spontaneous and agonist-induced oscillations in tension. Maximal tension development and cumulative dose response curves with catecholamines were equivalent in vessels tested with and without prior treatment with the beta-adrenergic receptor antagonist propranolol (10 5 m o l ' l - 1 ; five vessels tested in one snake). No decrease in tension was observed in any vessel during cumulative (10- l ~ mol. 1-1) catecholamine exposure (n = 8). High potassium (80 mmol-1-1) produced contractions similar in magnitude and pattern to cat-

echolamines in most vessels except those segments associated with the aortic arch junction (ADA, LAA and RAA), in which HI K was less effective. HI K + produced significantly more tension than either catecholamine in the PPV and M P V (Fig. la-c). Peptide hormones A N G II and AVT were weaker arterial agonists than catecholamines (Fig. ld, e). However, A N G I! was equipotent to catecholamines and HI K + in veins. The efficacy of A N G II in arteries decreased caudally (Fig. ld), opposite to that observed for catecholamines and HI K All vessels were tachyphylactic to A N G II at high (>10-Tmol-1-1) concentrations. Incubation with phentolamine (10- 5 tool" 1-1) did not appear to modify the maximum tension or potency of A N G II in the vessels tested (CA, PPV, JV, MPV; n = 4 snakes). Acclimation to hypergravity did not significantly affect any vessel except M D A where it reduced A N G II tension from 102 + 26 (n = 4 ) to 32_+ 12 (n = 4 ) m g . m g -1 wet weight (P < 0.05). A similar trend between A N G II-stimulated tension in C O N T and HI-G PDA was observed, although it was not statistically significant (119 _+ 55 vs 27 + 15; 0.10 > P > 0.05). AVT produced significantly greater tension in the aortic arches and ADA compared to other vessels (Fig. le). Maximal AVT-generated tension was transient, and it was followed by a decrease in tension to pre-AVT levels. This tachyphylaxis was observed in all vessels at 10- 7 mol- 1-1 AVT. ATP and histamine (10-5 tool" 1-1) contracted both resting and precontracted arteries and veins. ATP responses were vessel specific, and the pattern of stimulation was similar to that exhibited by catechotamines and HI K (Fig. if). All vessels were tachyphylactic to ATP and tension returned to pre-ATP levels within 30 rain of continuous ATP exposure. Histamine was a weak agonist ( < 1 5 0 t u g ' r a g -1 wet weight) in all vessels except PDA (473 _+ 203 m g - m g - 1 wet weight; Fig. lg).

Sensitivity The pD2s demonstrate vessel-specific differences in sensitivity (Table 1). In general, variability of pDas for a single agonist was approximately one order of magnitude between all vessels. Significant differences in pD2s were observed primarily between arteries and veins. The M D A and P P V were consistently the least sensitive vessels. In contrast, anterior vessels, CA, aortic arch vessels and JV, were significantly more sensitive to catecholamines than MDA, P D A and PPV. Few significant differences were observed between C O N T and H I - G snakes. Sensitivity of CA to A N G II was significantly reduced in HI-G snakes (pD2s: 7.04 _+ 0.15 vs 6.56 _+ 0.10). Sensitivity of the P D A to N E P I was significantly reduced in HI-G snakes (pD2s: 6.53 _+ 0.13 vs 5.95 + 0.06).

EPINEPHRINE
1600 '$ 1400 1200 1000
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1000
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ANGIOTENSIN II []
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RAA
ARTERIES VEINS

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LAA RAA ADA

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PDA PPV

NOREPINEPHRINE
[] [] LAA RAA * +

ARGININE VASOTOCIN
1000

+
600 600 400
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"~ 1600 9 ~ 1400 1200 "7 1000 800 600 400

200

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LAA RAP,
ARTERIES VEINS

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CA JV La~A RAA ADA HIGH POTASSIUM
"~ 1600 "$ 1400 1200 "7 1000 800
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PDA PPV

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ADENOSINE TRIPHOSPHATE ,
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ARTERIES VEINS

Fig. l a - g Anatomical distribution of maximal tension development in yellow rat snake, Elaphe obsoleta vessels. Tension in m g ' m g - ~ wet weight. Values = mean + SE: a epinephrine, 10 4mo1.1-1; b norepinephrine, 10 4mo1-1-1; c high potassium, 80mmo1"1-1; d angiotensin II, 3 x 10-6 m o l - l - 1 ; e arginine vasotocin, 10-6m01.1-1; f adenosine triphosphate, 10 S m o l ' l - 1 ; g histamine, 10 - s mol" 1- 1. + or * = significant difference between vessels with common symbol. + in le = significant difference between left aorticarch and all other arteries. Comparisons between arteries and veinswere not made. Note: scaling of ordinate varies with agonist. Vessel abbreviations: ADA, anterior dorsal aorta; CA, carotid artery; JV, jugular vein, LAA, left aortic arch, MDA, midbody dorsal aorta; MPV, midbody portal vein, PDA, posterior dorsal aorta; PPV, posterior portal vein; RAA, right aortic arch. n = 4-8 snakes for each agonist and vessel

.~' 600
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CA LAA
MDA MPV PDA PPV

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522

D.J. Conklin et al.: Reactivity of snake blood vessels in vitro

ACETYLCHOLINE 100 []
[]

Relaxation
Relaxations were agonist and vessel specific (Fig. 2). SNP produced significantly greater relaxation in most vessels than either ACH or ANP. ACH and SNP produced significantly greater relaxations than ANP in CA, JV and MPV. The magnitude of relaxations for most vessels was SNP > ACH > ANP; for CA, JV and MPV the order was SNP = ACH > > ANP. Average percent relaxation for all vessels with SNP and ACH were significantly correlated (r = 0.71; P < 0.05). No significant differences between C O N T and HI-G vessel relaxation were observed. Addition of indomethacin (10- s mol" 1-1) to precontracted veins generally decreased tension, but it did not affect the ACH-induced relaxation in JV (n = 4) or MPV (n = 2). Methylene blue (10 -5 mol" 1-1) further increased tension in precontracted veins, doubling it in two JV. Methylene blue partially blocked (approx. 50%) the ACH-induced relaxation of precontracted JV (n = 4) although it did not affect ACH-induced relaxation in MPV (n = 2). Peptides, ANG II, ANP and BK, were ineffective in el relaxation of precontracted vessels. ANG II (10- 7 mol- 1-1) did not relax any precontracted vessels (n = 5). ANP ( 1 0 - S m o l ' l 1) did not reduce tension more than 10% in 96% of all rings tested, and tension actually increased ( < 6 % ) in 17% of vessel rings. Similar to ANP, BK (10-v mol-1- x) did not reduce tension more than 10% in any precontracted vessel (n = 6). BK did produce a few small contractions ( <50 mg) in some vessels (n = 3). In contrast, ACH produced contractions in only 6% of precontracted vessels, while SNP did not increase tension in any vessels.

RAA
ARTERIES

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vEINs

2O-

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CA JV

LAA RAA ADA

MDA MPV

PDA PPV

SODIUM NITROPRUSSIDE
150 125

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50

25
0

b CA JV LAA RAA ADA MDA MPV

E~

PDA PPV

Fig. 2a,b Percent relaxation of catecholamine-precontracted yellow rat snake, Elaphe obsoleta, vessels: a acetylcholine, 10 . 4 tool' 1-1; b sodium nitroprusside, 10 . 4 mol-1 1. Values = m e a n SE. Vessel abbreviations as in Fig. 1. n = 4 8 snakes for each vessel

Discussion

Arboreal and semi-arboreal snakes exhibit cardiovascular traits that maintain systemic blood pressure and vital organ flow when challenged by posture-induced gravity stresses (Seymour and Lillywhite 1976;
Table 1 Sensitivity (pD2s) of yellow rat snake (Elaphe obsoleta) vessels to angiotensin II (ANGII), arginine vasotocin (AVT), epinephrine (EPI) a n d norepinephrine (NEPI). pD2 = log of effective c o n c e n t r a t i o n producing halfmaximal response (ECs0). Vessel abbreviations: see Fig I. Values = m e a n SE. (n) = n u m b e r of snakes

Lillywhite and Gallagher 1985; Lillywhite 1987). When yellow rat snakes are challenged by 45 ~ head-up tilt, blood flow to anterior tissues including brain, heart, and lungs is maintained, while blood flow is reduced

VESSEL ANG II ARTERIES'

CA LAA RAA ADA MDA PDA 7.04 6.50 7.83 6.56 6.70 6.82 0.15 0.10 _+ 0.86 _+ 0.16 0.12 0.24 (3) (7) abe (2) (7) ae
(7) fg

AVT
8.06 7.48 7.64 7.39 8.12 8.47 0.67 0.07 (1) 0.06 0.69 0.84 (4) (6) (6) (4) (3)

EPI
7.04 7.25 7.27 6.97 6.52 6.54 0.37 0.07 _+ 0.40 0.12 0.17 0.16 (8)
(7) abe

NEPI
6.88 7.07 7.06 6.72 6.02 6.53 0.26 0.19 0.39 0.14 _+ 0.12 _+ 0.13 (8) ab
(8) cde

(4)

(4) (7) ~c (8) b (8)"

(4) (8) ~ (8) "of (4)

VEINS
JV MPV PPV 7.30 + 0.13 (7) TM 7.30 0.19 (7) bg 6.80 _+ 0.30 (6) ce 8.26 0.52 (4) 8.37 0.40 (5) 7.72 _+ 0.80 (2) 6.82 _+ 0.19 (8) a 6.30 0.08 (7) ~ 6.04 + 0.08 (7) "ha 7.16 _+ 0.19 (8)fgh 6.19 0.11 (8) bdg 6.15 + 0.12 (8) ~h

abcdefgh significant difference between vessels with same letter for a c o m m o n agonist (P _< 0.05)

D.J. Conklin et al.: Reactivityof snake blood vessels in vitro significantly in visceral organs, posterior muscle, and posterior skin (Lillywhite and Gallagher 1985). Rapid adjustments in peripheral vascular resistance and venous tone during head-up tilt prevents blood pooling and promotes maintenance of venous return and cephalic blood flow. There is relatively little information concerning the mechanisms that mediate these changes in peripheral vascular resistance and venous tone.

523

posterior arteries is involved in regulating peripheral resistance during postural stress (Lillywhite 1987; Donald and Lillywhite 1988; Lillywhite and Donald 1994). However, it was observed that anterior vessels were ~ 3 - 1 0 times more sensitive to catecholamines than posterior vessels (Table 1). The significance of this difference and its mechanism are unclear. Vascular sensitivity may be directly related to neuronal density observed in different anatomical locations. Cocaine, which blocks neuronal uptake of catecholamines, significantly increases the sensitivity of the dorsal aorta Sympathetic mechanisms to catecholamines in the Brazilian snake, Bothrops The sympathetic nervous system is extremely impor- jararaca (Yamanouye et al. 1992). Alternatively, antant for maintenance of blood pressure during ortho- terior vessels may be more sensitive to circulating catstasis and postural adjustments in mammals (Rowell echolamines than are posterior vessels. A distinction 1993). In anesthetized cats, for example, the sympath- between humoral and neuronal adrenergic receptors olytic drugs bretylium and guanethidine completely has been observed in the trout vasculature (Xu and block the restoration of systemic arterial pressure dur- Olson 1993). Functionally, this may be important ing 45 ~ head-up tilt (Nolan and Bentley 1978). High- during head-down posture as a mechanism to density sympathetic innervafion in blood vessels of the prevent posturally-induced, cephalic edema, or in hemgiraffe are likely representative of adaptive traits in- orrhage or other hypotensive states when plasma volved in regulating hemodynamics of long vessels sub- catecholamine concentrations would be expected to jected to large hydrostatic gradients and high arterial increase. Active regulation of venous tone is presumably impressures (Nilsson et al. 1988). Scansorial and arboreal snakes as well as other long or tall terrestrial verte- portant for maintaining return of venous blood to the brates are potentially subjected to similar problems heart during head-up posture or climbing in snakes. In addition to dense sympathetic innervation of arterial and presumably have evolved parallel adaptations. Studies on isolated vessel strips indicate that both vessels in E. obsoleta, equally dense innervation occurs adrenergic and cholinergic vasomotor fibers innervate in posterior portal veins (Donald and Lillywhite 1988). the major arteries of lizards, and it has been suggested Central venous pressure increases following in vivo that adrenergic innervation is important in the main- administration of catecholamines and remains stable tenance of reptilian vascular tone (Burnstock 1969; during graded hemorrhage (Lillywhite and Smith 1981; Kirby and Burnstock 1969). Sympathetic contraction Lillywhite 1987). These conditions presumably reflect of posterior blood vessels has been implicated as an an adrenergically-mediated increase of venous tone. important mechanism for maintenance of arterial The posterior portal vein produces considerable tenblood pressures in snakes (Lillywhite and Seymour sion in response to catecholamine stimulation in vitro, 1978; Lillywhite and Gallagher 1985; Lillywhite 1987). so the dense innervation of veins probably reflects its Subsequent findings have demonstrated the presence of role in active regulation of venous tone and capacity. dense adrenergic innervation in E. obsoleta as well as However, anterior veins have far less sympathetic other species of snakes (Donald and Lillywhite 1988; innervation than do posterior veins in E. obsoleta, yet Lillywhite and Donald 1994), Innervation density in E. in vitro tension of veins produced by catecholamines is obsoleta appears to be greater than in other reptiles, similar in the two regions. Several factors might acincluding other aquatic and terrestrial snakes, and sug- count for this including differences in receptor accessigests that adrenergic mechanisms are important for bility, affinity or population. In any case, control of hemodynamic regulation during postural and gravi- tone in the jugular vein would be important in normal tational stress. Sympathetic fibers are most dense in physiological regulation of venous return and possibly posterior vasculature, with comparatively fewer fibers plays a significant role during head-down postures, in the anterior vessels of E. obsoleta (Donald and or other hemodynamic perturbations such as hemorrhage. Lillywhite 1988). Although specific adrenoceptors were not characterSystemic vessel responses to catecholamine stimulation in vitro mirror the pattern of vascular adrenergic ized in this study, the observed catecholamine-meinnervation along the length of the yellow rat snake diated responses are similar to those identified in the (Donald and Lillywhite 1988). Tension development in Brazilian snake Bothrops jararaca (Yamanouye and response to catecholamines is weakest in the most Picarelli 1992; Yamanouye et al. 1992). fi-adrenergic anterior artery (CA), and strongest in the PDA receptors appear to be absent in the systemic arteries of (Fig. la, b). This observation supports the conclusion the Brazilian snake (Yamanouye et al. 1992). Catthat increased density of sympathetic innervation in echolamine dose-response curves were unaffected by

524 propranolol (a non-specific fi-receptor antagonist), and no decrease or transient drop in tension during exposure was observed (Yamanouye et al. 1992). This pattern was found in both arteries and veins of E. obsoleta and suggests that fi-mediated vasodilatory responses are also absent in the large vessels of the systemic circulation. However, fi-adrenergic-mediated vasodilation is involved in the neural regulation of pulmonary vasculature in E. obsoleta (Donald et al. 1990a) and in the aquatic snake Acrochordus 9ranulatus (Lillywhite and Donald 1989). In both species neural stimulation elicits both cholinergic vasoconstriction and adrenergic vasodilation of the pulmonary vasculature. Vasodilator fi-receptors may be present in the systemic vasculature of the sea snake, Acalyptophis peronii, insofar as EPI produces a dose-dependent decrease of blood pressure in vivo (Lillywhite and Pough 1983). In comparison, the sensitivity (pD2) of E. obsoleta isolated dorsal aorta (MDA, 6.02; PDA, 6.53) to N E P I is similar to pD2s obtained from dorsal aorta of the cobra, Naja naja ( ~ 5.9), rat snake, Ptyas korros [ ~ 6.0; Yung and Chiu (1985)] and Brazilian snake, Bothrops jararaca [6.63; Yamanouye et al. (1992)]. These data suggest that snakes share a structurally similar eadrenoceptor in the dorsal aorta. Few data exist for comparison between other vessels or anatomical locations. Local factors/hormones Endocrine, autocrine and paracrine mechanisms are undoubtedly as important for hemodynamic regulation of peripheral vasculature in reptiles as they are in mammals. These factors have received limited attention in reptiles, however (Chiu et al. 1986; Kirby and Burnstock 1969; Knight and Burnstock 1993; Miller and Vanhoutte 1986; Reite 1970). Present findings suggest that putative hormones and local factors are involved in regulating vascular tone. A renin-angiotensin system is involved to varying degrees in cardiovascular function of most vertebrates and appears to be present in snakes (Gervitz et al. 1987; Lazari et al. 1994). Arterial reactivity to ANG II decreases along the length of the body in a manner opposite to the pattern of catecholamine and HI K response (cf. Fig. la-d). Thus, ANG II stimulates significantly greater tension in the carotid artery when compared to mid- and posterior aortic segments. This pattern may be due to an increased concentration of ANG II receptors in the carotid artery. ANG II contracts the carotid artery, but not the dorsal aorta, of the arboreal snake, Ptyas korros (Yung and Chiu 1985). Furthermore, carotid arterial blood pressure is dosedependently increased by Aspl-ileCANG I1 in Bothropsjararaca in vivo (Breno and Picarelli 1992). These data collectively imply a specific role for ANG II in the control of carotid flow in climbing snakes.

D.J. Conklin et al.: Reactivityof snake blood vessels in vitro The ANG II vascular response in isolated vessels of the yellow rat snake appears independent of catecholamine interaction, unlike the situation found in the dogfish shark (Opdyke et al. 1982). ANG II produces catecholamine-independent contractions in both arteries and veins of E. obsoleta, as in the isolated dorsal aorta of the cobra, Naja naja (Yung and Chiu 1985), and turtle aorta (Stephens 1984). These data suggest there is a functional separation of ANG II and catecholamine receptors in snakes in vitro. However, approximately 47% of an Aspl-ileCANG II-mediated increase in arterial pressure in Bothrops jararaca was blocked by preinfusion with the ~-adrenergic antagonist, phenoxybenzamine (Breno and Picarelli 1992). ANG II relaxes precontracted arteries and veins of several vertebrate species including fish, birds and mammals, but not bullfrogs (Webb 1982; Yamaguchi and Nishimura 1988; Conklin and Olson 1994b). Attempts to relax precontracted arteries and veins of yellow rat snake with ANG II in this study were unsuccessful. This may be due to the heterologous peptide used in this study (salmon ANG II), although human ANG II is equally effective as salmon ANG II in producing relaxation in isolated trout vessels (D.J. Conklin, unpublished observation). More likely, the receptors mediating relaxation to ANG II are not present in snakes. The peptide hormone AVT produces vessel-specific contraction, essentially limited to the aortic arches and the anterior dorsal aorta. Contraction of these vessels may preferentially increase resistance to posterior systemic blood flow, and thereby increase either cephalic or pulmonary blood flow (Lillywhite and Donald 1994). The vessel-specific action of AVT is also observed in trout vessels where AVT contracts arteries, anterior cardinal vein and ductus Cuvier but has little effect on posterior cardinal and intestinal veins (Conklin and Olson 1994a). However, AVT and other neurohypophysial analogs produce a potent vasodepressor response in vivo in the rat snake, Ptyas korros (Chan 1977). Furthermore, blood extracts from the Brazilian snake, Bothrops jararaca, that contain an AVT-like peptide produce antidiuresis and potent hypotension in the rat (Silveira et al. 1992). It is not clear whether this hypotensive extract is related to the generally weak contractile action or some other effect of AVT in yellow rat snake blood vessels in vitro. The pattern of tension produced in all vessels in response to HI K + is similar to that produced by catecholamines. The HI K + response is far greater in posterior portal vein than in the jugular or midbody portal vein, and is comparable to that observed in the posterior dorsal aorta (Fig. lc). This finding suggests that posterior systemic vessels may possess more voltage-operated channels than are characteristic of anterior vessels. These data also complement an earlier finding that a HI K + response may not be equivalent to

D.J. Conklin et al.: Reactivity of snake blood vessels in vitro

525

the maximal agonist-induced response in trout blood vessels (Conklin and Olson 1994a). ATP and histamine produced contraction patterns similar to that produced by catecholamines. ATP and histamine contract mammalian and reptilian vascular smooth muscle, respectively, at high doses comparable to those used here (10 -s tool-l-l; Dezsi et al. 1990; Miller and Vanhoutte 1986). ATP and histamine can produce relaxation through stimulation of the endothelium in mammals (Dezsi et al, 1990), and intravenous histamine reduces arterial pressure in the European common viper, Vipera berus (Reite 1970). However, no relaxations with ATP or histamine were observed in this study. The role of these putative local factors and their potential dual responses in control of vascular tone in snakes still needs to be elucidated. Only a few agonists relaxed precontracted vessels in this study. The degree of relaxation produced by ACH and SNP was significantly correlated among all vessels tested (r = 0.71, P < 0.05). This suggests that ACH and SNP may be acting through a common mechanism, possibly nitric oxide, to stimulate soluble guanylyl cyclase. Sodium nitroprusside forms nitric oxide spontaneously (Ignarro et at. 1981), and ACH stimulates endothelium-dependent relaxation in many vertebrates (Miller and Vanhoutte 1986), including snakes, via the release of nitric oxide (Knight and Burnstock 1993). Methylene blue, a scavenger of nitric oxide and inhibitor of guanylyl cyclase activation, blocks ACHinduced relaxations in jugular veins by approximately 50%, but not in the midbody portal vein of E. obsoleta. Methylene blue also inhibits A23187, calcium ionophore, endothelial-dependent relaxations in cayman, Caiman crocodilus, aorta (Miller and Vanhoutte 1986). It is not clear what part of the ACH-induced relaxation methylene blue is affecting in the JV and not affecting in the MPV. Prostaglandins do not appear to mediate the ACHinduced relaxation in reptiles. Preincubation with indomethacin (a cyclo-oxygenase inhibitor that blocks prostaglandin production) does not inhibit ACH-induced relaxation in precontracted veins tested herein. Similarly, ACH-induced relaxation in the aorta of the cayman is not blocked by meclofenamate (Miller and Vanhoutte 1986). These results suggest that a non-prostanoid substance maybe involved in ACH-induced relaxation in yellow rat snake veins. The physiological significance of the ACH-induced relaxation is not clear, but it might contribute to regulation of overall vascular tone and especially posterior-to-anterior shunting of blood flow during circumstances of upright posture (Lillywhite and Donald 1988). Understanding the role of the endothelium, nitric oxide and other substances in control of vascular tone in snakes will require further investigation. Peptide hormones (ANP, BK) are generally ineffective in producing relaxations of precontracted vessels. Surprisingly, both ANP and BK produced a few small

contractions in precontracted vessels instead of relaxation. The lack of ANP- or BK-mediated relaxation may be due to the heterologous nature of the peptides (mammalian forms), although rat ANP stimulates relaxation in precontracted trout vasculature (Olson and Meisheri 1989). However, bradykinin generated from plasma of the reticulated python, Python reticulatus, differs significantly in sequence from the mammalian BK used in this study (Conlon and Lance 1994).

Acclimation to hypergravity Interestingly, two of the three significant changes observed in the acclimated snakes are related to ANG II and they suggest an important role for the renin-angiotensin system in adaptation to elevated + Gz. Results of the acclimation experiment suggest that sensitivity and efficacy of ANG II responses can be modified to some extent in various vessels. Insofar as increased levels of ANG II are expected during increased G stress in mammals (Oparil et al. 1970; Harrison et al. 1986), reduced arterial sensitivity, especially in the carotid artery, might act in a compensatory manner to assist the maintenance of arterial blood flow during head-up posture or Gz stress. Clearly, more information on the cardiovascular role of the renin-angiotensin system in snakes is needed (Gervitz et al. 1987; Lazari et al. 1994). The lack of prominent or widespread changes between vessels of acclimated and control snakes is conceivably a result of limited sample size. A few changes were observed, and possibly more would be revealed either by examination of more animals or employment of increased + Gz. Moreover, species with lesser genetic adaptation to gravitational stress than is present in E. obsoIeta (e.g., aquatic or amphibious species) might exhibit more plasticity in vascular reactivity. On the other hand, vascular adaptation to gravity stress is quite possibly genetically fixed with little physiological plasticity. Clearly, this is an area for further research.
Acknowledgements The authors thank C. Wade, J. Greenleaf and two anonymous reviewers for suggestions concerning the manuscript. C. Gordon helped with the manuscript preparation. This work was supported by NSF grant no. IBN 91-05247 to K.R.O., NASA grant 199-14-12-04 to A.R.H., and a National Research Council Senior Research Associateship to H.B.L. All experiments comply with the guidelines in "Principles of animal care", publication No. 86-23 of the National Institutes of Health, revised t985.

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