En el ruedo

Onset of h ib ern atio n is accom panied by a decline in heart r a t e

and consequent decrease in systolic and diastolic b lo o d

pressure. Despite this fall, the mean arterial p ressu re r e m a i n s within the range observed in the normal conscious animals, b y an increase in perip h eral resistance (Lyman & O'Brien, 1 9 6 0 ;

Bullard & Funkhouser, 1962; Kirkebo, 1968; Wells, 1 9 7 1 ;

A lbert & Panuska, 1978; Zatzman, 1984). Lyman and O'Brien (1963) suggested that, although some of the observed c h a n g e s

in the blood flow were attributable to a t e m p e r a t u r e -

d e p en d e n t increase in blood viscosity, an increase in v a s c u l a r tone was also a contributing factor. Maclean (1981) r e p o r t e d that several characteristics of blood of hibernators combined to make it unlikely that an increase in blood viscosity was a major con trib u to r to the observed increase in the p e r i p h e r a l resistance. The latter argum ent was further supported by work

of Miller and colleagues (1986) who showed t h a t

resp o n siv en e ss to no rad ren alin e (NA) was increased in r e n a l vessels of hib ern atin g compared with n o n - h i b e r n a t i n g

w oodchucks Marmota M o n a x . This adaptive response w a s

specific and led to the hypothesis that changes in v a s c u l a r responsiveness contribute to the regional control of blood flo w in hibernating animals.

During h ib ern atio n renal function is reduced to 10 % o f its eutherm ic level in many hibernating species. The m a r m o t , and the bear d e m o n strate an increase in renal v a s c u l a r

resistance indicated by increased Alteration fraction (Brown e t

The extensive influence of sym pathetic p e r i v a s c u l a r nerves on the regional control of blood flow in m any m a m m a l s

is well established. Evidence has accum ulated that NA,

adenosine 5'-triphosphate (ATP) and n e u ro p ep tid e-Y (NPY) a c t

as c o -tra n sm itters in p erivascular sym pathetic n e r v e s

(Burnstock, 1990d), The control exerted by each of t h e s e

substances differs considerably in different blood vessels a n d species (Burnstock, 1995). Sym pathetic n e u ro tra n sm is sio n h a s been described in the renal vasculature of rat, rabbit, dog a n d hum an (Petrovic et a l , 1988; Kooner et a l , 1988; Luff et al,

1991). However, the inv o lv em en t of the sym pathetic n e r v o u s system on the regional control of renal blood flow in h a m s t e r has not been described, nor has its role in the local control o f vascular tone during hibernation.

The p resent study investigates factors influencing local control of vascular tone in age matched controls (controls), cold controls and hibernating golden hamsters. To this end, is o l a t e d renal arterial rings were used to evaluate the s y m p a t h e t i c n e u ro tra n sm is sio n and assess a d r e n e r g i c - p u r i n e r g i c p e riv a s c u la r c o -tra n sm issio n .

M e t h o d s

A n i m a l tre a tm en t

Syrian or golden ham sters {Mesocricetus a u r a r t u s ’, 1 2 5 - 150 g) were used, since they are perm issive h ib ern ato rs a n d

directly influenced by external en v iro n m en tal c o n d itio n s

(Morrison, 1960). H ibernation in this species can be i n d u c e d by m an ip u latin g photoperiods or te m p era tu re and c r e a t i n g p s e u d o -w in te r conditions in the laboratory at any time of t h e year (Hoffman, 1965). Male ham sters were caged in groups o f

five in a refrig era ted incubator (Leec Ltd, Nottingham, UK)

initially set at 20° C and with the lig h t/d ark period set at 8 :1 6

h light. The period of light was reduced by 30 min d a ily ,

to g eth er with a gradual tem p era tu re reduction to reach t h e

final condition of 2 h light each day and an a m b i e n t

te m p e ra tu re of 9° C. This climatization procedure took 7 - 1 0 d a y s .

The animals were then tran sferred to individual cages in a cold room which was set at 5° C with 2 h of light per day f o r 14 weeks with food and water ad lib i t u m . Inspection of t h e

animals outside the designated light period was o n ly

p e rfo rm ed under a 10 watt red photographic light. To c h e c k w h eth er the animals were hibernating, sawdust was s p r i n k l e d

on their backs (which would waken a sleeping but not a

h ib ern atin g ham ster). Animals were in hibernation for 4

weeks, with periodic arousals, but were sacrificed only after a deep hibernation bout of 1-4 days. Animals which u n d e r w e n t

similar treatm ent but did not hibernate were used as cold

controls. Age (3 m onths) and sex m atched animals kept a t

room tem perature were used as controls.

In vitro pha rm aco lo gy

The ham sters were sacrificed by an overdose of c a r b o n

im m ed iately after asphyxiation. Renal arteries were d i s s e c t e d

and cleaned of excess tissue. Ring segm ents of 3-4 mm in

length were cut and m ounted in 5 ml organ baths c o n ta in in g

(mM): NaCl 133, KCl 4.7, NaH2P0 4 1.35, NaHCOg 16.3, MgS0 4

0.61, CaCl2 2.52 and glucose 7.8, gassed with 95% O2, 5% CO2 a t 3 7 0 C. Rings were left to equilibrate for 60-90 min under a resting tension of 1 g, which was taken as the basal tone of t h e

p re p a ra tio n and was m ain tain ed thro u g h o u t t h e

ex p erim e n tatio n . Isometric tension was recorded with a G rass FT03C tran sd u cer and displayed on a Grass i n k - w r i t i n g polygraph (model 79).

In renal arterial rings, tran sm u ral nerve s t i m u l a t i o n

(TNS) was achieved by passing a current betw een tw o

electrodes parallel to the arterial rings. The p a ra m eters f o r TNS( 80 V, 0.1 ms, 4-64 Hz, for 1 s) were selected, since t h e y

evoke both adrenergic and purinergic com ponents o f

p e riv a sc u la r sym pathetic n e u ro tra n sm is sio n in other s i m il a r p rep a ratio n s (Karoon et a l , 1995). R eproducible v a s c u l a r responses were obtained with 1 s duration of TNS. P r a z o s in and a ,p -m e th y le n e ATP were added to the bath 20 min p r i o r to construction of fre q u e n c y -re sp o n s e curves. D e s e n s itis a tio n

to a ,p -m e th y le n e ATP was confirm ed by no response to a

rep eated dose of the antagonist. Cum ulative c o n c e n t r a t i o n - response curves to NA and ATP were established.

Contractile responses in renal arterial prep aratio n s, w e r e e v aluated as an increase in tension (g). In all p r e p a r a t i o n s

contractile response to 120 mM of KCl was measured.

Drugs used

N o rad ren alin e bitartrate, adenosine 5 '-trip h o sp h a te d i s o d i u m salt, prazosin, a ,p -m e th y le n e ATP, and tetrodotoxin, w e r e

obtained from Sigma Chemical Company (Poole, E ngland).

M onoam ines were dissolved in ascorbic acid (0.1 mM); all other drugs were dissolved in distilled water.

Data Analysis

All data are expressed as means ± S . E. M. The p Ü2 values w e r e calculated as the negative log of the concentration required to

produce 50% of maximal response. Statistical analysis w a s

p erfo rm ed using analysis of variance and paired or u n p a i r e d Student's t test as appropriate for individual points and o n e way analysis of variance (ANOVA) for the entire curve, u s in g 'Instat' program m e. A probability value of P less than 0 .0 5 was considered statistically significant .

R e s u l t s

A n i m a l tr e a tm en t

Ham sters started hibernating after 10 weeks in a cold r o o m and were allowed to hibernate for a further 8 weeks. D u rin g this time, a hibernation rate of 42% with 3-5 days bouts o f deep h ib ern atio n was reached. There was a significant w e i g h t loss in both hibernating and cold controls com pared with t h e

control group (Table 3.1). Cheek pouch and rectal b o d y

animals com pared with both cold control and control g r o u p s (Table 3.1).

P h a r m a c o l o g y

In golden ham ster renal arterial rings, TNS (4-64 Hz) e v o k e d f r e q u e n c y - d e p e n d e n t contractile responses of d i f f e r e n t m agnitude in the three experim ental groups (Fig. 3.1). T h e s e responses were abolished by application of tetrodotoxin (1 p M ) ,

thus revealing their neural origin. In the eutherm ic s t a t e

(control group), the contractile responses to TNS was o n ly

revealed at higher frequencies of stimulation; during cold

exposure (cold control group) these responses w e r e

significantly greater. However, in the hib ern atin g g r o u p

contractile responses to TNS was also shown at l o w e r

frequencies and were significantly higher than either control or cold control groups at all frequencies tested (Figs. 3.1 & 3.2). A t 64 Hz vascular responses were 0.27 ± 0.06 (?2=5), 0.10 ± 0 . 0 2 (n=7) and 0.05 ± 0 .0 1 (n=5) g i n hibernating, cold control a n d controls, respectively (Fig. 3.2).

In the presence of 1 pM prazosin, vascular responses to

TNS were reduced in all three groups tested (Fig. 3.3).

Fu rth erm o re, exposure of prep aratio n s to the d e s e n s i ti s i n g agonist of P2 X -p u rin o cep to r a ,p - m e th y le n e ATP (12 pM), produced rapid, transient contractions in all the p r e p a r a t i o n s tested, after which the residual com ponent of TNS was n o t significantly reduced (Fig. 3.3).

In hibernating animals the no rad ren erg ic com ponent o f

p eriv a sc u la r sym pathetic n eu ro tra n sm issio n was greater t h a n

in both control and cold control groups with s t a tis tic a l

significance being reached at 8, 16, 32, 50 and 64 Hz. In cold control animals the n o radrenergic com ponent of p e r i v a s c u l a r sy m pathetic n e u ro tra n sm issio n was greater than in c o n tr o l animals at 50 and 64 Hz.

A pplication of exogenous NA (0.1-100 |iM), to the r e n a l arteries, evoked co n centration-dependent contractile r e s p o n s e s which were significantly greater in hibernating com pared w i t h control animals (Fig. 3.4a). However, there was no s ig n if ic a n t difference betw een hibernating and cold control animals. N A -

evoked maxim al responses were significantly greater in

hib ern atin g animals com pared with eontrol animals, but d i d

not differ betw een hibernating and cold controls a n im a l s

(Table 3.2). No significant difference was shown among the pD2 values of the three experimental groups (Table 3.3).

V asoconstrictor responses of the renal artery to ATP ( 1 -

3000 |iM), were not significantly altered in either t h e

h ib ern atin g animals or cold controls com pared with c o n tr o l animals (Fig. 3.4b). Similarly, the maximal contractile r e s p o n s e and the pD2 values did not differ significantly among the t h r e e experimental groups (Tables 3.2 & 3.3).

Contractile tension developed by 120 mM KCl, did n o t differ significantly among the three groups of animals t e s t e d (Table 3.2).