El ensayo: torería en la expresión y en la forma

2 .1 .1 A r t e r ia l rin g p r e p a r a tio n

The following method applies to p rep a ratio n of rat ta il artery for the studies in chapters 3 and 4, rat femoral artery in chapter 7 and hamster renal artery in chapter 5. Animals w e r e sacrifised by CO2 asphyxiation, an overdose of p e n t o b a r b i t o n e

or ether. Selected arteries were dissected im m ed iately a n d

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

length, w ere cut and m ounted horizontally under is o m e tr ic

condition in 5-ml organ bath by inserting a fine tungsten w i r e (125 p m diam eter) through the lumen of the vessel ring a n d

anchoring it to a stationary support (according to Beven &

Osher 1972). Another wire, similarly inserted, was c o n n e c te d by silk thread to a Grass D7 force disp lacem en t tran sd u cer a n d resp o n ses were recorded on a Grass ink-w riting p o l y g r a p h (Quincy, MA, USA). Arterial rings were placed under a r e s t i n g tension of Ig and allowed to equilibrate for 60-90 minutes, in B u lb rin g -m o d ifie d Krebs solution of the following c o m p o s itio n (mM): N a d 133, KCl 4.7, N a H 2 P 0 4 L52, NaHCOs 16.3, M gS 0 4

0.61, glucose 7.8, CaCl2 2.52. The Krebs solution w a s

m aintained at 37 and aerated with a m ixture of 95% O2 a n d

59&CC%L

2 .1 .2 T r a n s m u r a l nerve stim u la tio n o f a r te r ia l rings

T ran sm u ra l nerve stim ulation (TNS) was achieved b y

passing a current across the preparation betw een two t u n g s t e n wires that were placed parallel to the arterial ring and acted as two electrodes. P aram eters for TNS were selected in order to

activate both n o rad ren erg ic and purinergic com ponents o f p e riv a sc u la r sym pathetic n e u ro tran sm issio n . R e p r o d u c ib le v ascu lar responses were obtained at a frequency of 4-64 Hz, supram aximal voltage of 65-80 V, pulse duration of 0.1 ms f o r

1 s.

2 .1 .3 I s o la te d m e s e n te r ic a r te r ia l bed p r e p a r a tio n

The following m ethod applies to p rep aratio n of r a t

isolated m esenteric arterial bed for the studies in chapter 6. Rats were sacrificed by asphyxiation with ether. The a b d o m e n

was opened and the superior m esenteric artery exposed a n d

cannulated with a hypoderm ic needle. The superior m e s e n t e r i c vein was severed, the gut dissected away and the p r e p a r a t i o n m o u n ted on a stain-less steel grid ( 7 X 5 cm) in a h u m i d ch am b er (cu sto m -m ad e at University College London, UK). T h e p re p a ra tio n was perfused at a constant flow rate of 5 m l / m i n

using a peristaltic pump (model 7554-30, C o le - P a r m e r

In stru m en t, Chicago, XL, USA). The perfusate was K rebs'

solution of the following composition (in millimolar q u a n titie s ) : N a d 133, KCl 4.7, N a H 2 P 0 4 1.52, NaHCOs 16.3, M g S 0 4 0.61, glucose 7.8, CaCl2 2.52. The Krebs solution was m aintained a t

37 °C and aerated with a m ixture of 95% 0 2 and 5% CÜ2.

Responses were measured as changes in perfusion pressure (in m illim eters of m ercury) with a pressure tran sd u cer ( m o d e l P23XL, V iggo-Spectram ed, Oxnard, CA, USA) on a side arm o f the perfusion cannula and recorded on a polygraph (model D7,

Grass In stru m en t, Quincy, MA, USA). Preparations w e r e

2 .1 .4 E lec tr ic a l field stim u la tio n of arterial beds

Electrical field stim ulation (BPS) were established b y

passing a current (4-32 Hz, 90 V and 1msec for 30 sec) a c ro ss the preparation between the metal needle and the wire grid o n which the preparation rested, which acted as two electrodes.

After a recovery period of 10 min, prep aratio n s w e r e repetitively electrically stimulated (32 Hz, 90 V and 1 msec f o r

5 sec) at 2-min intervals to produce trains of p h a s ic

constrictions. The effect of adenosine (10, 30 pM) on these w a s

further assesed by application of each concentration o f

adenosine to the p erfusate for 6 min, followed by a period o f

w ashout betw een the concentrations but with o n g o in g

electrical field stimulation.

2. 1. 5 A p p lica tio n o f agonists & antagonists

Full c o n c e n tra tio n -re s p o n se relationship w e r e constructed and pD 2 values (negative log of concentration o f agonist producing half of maximal response) or pD v5 v a lu e s (negative log of concentration of agonist producing response of 0.75 g of tension) expressed where required. Agonists prone to desensitise tissues were applied as a single c o n c e n tr a tio n . Increasing concentration of agonists were added c u m u l a t i v e l y when no desensitisation was seen to occur.

Potential antagonists were left in contact with t h e

p rep a ratio n under exam ination for a minim um of 20 m in ,

unless otherw ise stated. C o n cen tratio n -resp o n se or f r e q u e n c y - response relatio n sh ip s were then re-established.

For vascular bed preparations, drugs given as b o lu s

injections of a fixed volume (usually 50 or 100 pi) a r e

e x p re ssed either as the "applied concentration" ( t h e c o n cen tratio n (M) of the stock solution used, or as the "dose" (the num ber of moles (mol) within the bolus). W hen d r u g s w ere added to the perfusate reservoir the final c o n c e n tr a ti o n (M) of the solution is stated.

2 .1 .6 A p p lic a tio n o f V a so d ila to r agents

In order to examine vasodilatation, tone was applied to p re p a ra tio n s by the use of a suitable contractile agent t h a t caused a sustained, stable contraction. An EC50 c o n c e n tr a ti o n of this agent was generally used, unless otherwise stated. Once contractile tone reached a plateau vasodilatory responses w e r e exam ined in a cumulative fashion and expressed as p e r c e n t a g e relaxation of the raised tone.