INTRODUCTION

As mentioned in the general introduction, the cardiovascular changes associated with the alerting stage of the defence response are partly mediated by the attenuation of the baroreceptor reflex (Hilton, 1963; Coote, Hilton & Perez- Gonzalez, 1979) and facilitation of the chemoreceptor reflex, (Hilton & Joels, 1965; Silva-Carvalho et al. 1993). Evidence has accumulated to suggest that these responses are mediated by synaptic interactions at the level of the NTS (McAllen 1976; Mifflin et al. 1988; Silva-Carvalho et al. 1993). Indeed,

suppression of the baroreceptor reflex is specifically mediated by activation of GABA^ receptors located within the NTS (Jordan et al. 1988). Moreover, it appears that intrinsic NTS GABA-containing neurones mediate these effects (see Spyer, 1994 for review).

Preliminary studies in the cat have illustrated that the intravenous administration of adenosine A, antagonists, which acts both peripherally and within the CNS, can augment the baroreceptor reflex and attenuate the chemoreceptor reflex whilst purely peripherally acting adenosine antagonist are ineffective (Dawid- Milner et al. 1994). Thus, it seems reasonable to suggest that adenosine released at sites within the CNS would elicit influences on these reflexes resembling the changes in the reflex efficacy observed during HDA stimulation (see Silva- Carvalho et al. 1993). Further, this might imply that adenosine released in the CNS may play a role in the cardiovascular components of the defence response.

Indeed, Dawid-Milner et al. (1994) have demonstrated an involvement of adenosine Aj receptors in the pressor response evoked during stimulation of the HDA in the cat.

To test this hypothesis this present study examines the effect of intravenous administration of two very different adenosine receptor antagonists, 8- Cyclopentyl,-l,3,-dipropylxanthine (DPCPX) and 8-(p-

sulphophenyl)theophylline (8-SPT), on the cardiovascular changes associated with the defence response in rats. DPCPX is a potent adenosine receptor

antagonist with approximately 700 fold selectivity for the adenosine Aj receptor over the A2 receptor (Lohse, Klotz, Lindenbom-Fotinos, Reddington &

Schwabe, 1987). It is a highly lipophilic drug and as a consequence its action is not only confined to the peripheral nervous system, but has access to the CNS, through its ability to cross the blood brain barrier (Thompson, Haas &

Gahwiler, 1992). Unlike DPCPX, 8-SPT is a polar drug and as a result its action is confined to the peripheral nervous system and tissues (Daly, 1982). Therefore, a comparison of the influence of these antagonists on the HDA stimulation would determine whether adenosine is involved in this response in the rat and further, if any effect is mediated by peripheral or central adenosine A, receptors.

3.2. METHODS

Experiments were conducted on 32 male Sprague Dawley rats (300 -350g). In each animal arterial blood pressure, tracheal pressure, end tidal CO2 and ECG were recorded as described in Chapter 2. For these experiments the animals head was fixed in a stereotaxic head frame with the skull in a level position. In order to further characterise the hypothalamic defence response, hindlimb blood flow was measured in 3 animals. The left femoral artery was exposed and the paw ligated so that blood flow to hindlimb skeletal muscle was recorded predominantly. Blood flow was measured by placing an electromagnetic flow probe (1.5mm circumference) around the left femoral artery. The probe was connected to a square wave electromagnetic flowmeter (Carolina Medical Electronics Inc, Model 501)). A zero signal was obtained at regular intervals throughout the experiment by brief occlusion of the artery distal to the flow probe with a pair of small curved forceps. The flow probe was later calibrated in vitro using constant flow perfusion. Femoral blood flow (FBF) was displayed in CHART on a computer screen via a 1401/7/w.s (CED).

Drugs

The following drugs were used: DPCPX and 8-SPT (Research Biochemicals International) and Ethanol (BDH Chemicals).

3.3. PROTOCOL

Cumulative intravenous dose response curves to DPCPX and Ethanol

Control responses to HDA stimulation were characterised (see Chapter 2) prior to the administration of the first dose of DPCPX. In 7 animals each dose of DPCPX was administered slowly over a period of 5 min into the femoral vein and the cannula flushed with an equivalent volume of saline. Doses of DPCPX administered were 0.3, 0.7 and 2 mg kg‘* dissolved in final volumes of 0.03, 0.06 and 0.2 ml. Due to the insolubility of DPCPX in saline/aqueous base, DPCPX was dissolved in absolute ethanol. The effect of DPCPX on the defence response was tested 5 min after the completion of administration of each dose of the drug. Doses of DPCPX were administered at 12 min intervals, which was sufficient to allow pressure to return to pre-stimulus resting values.

Since DPCPX was dissolvediin absolute ethanol, which in itself could evoke changes in cardiovascular parameters, (Zhang, Abdel-Rahman & Wooles, 1989; Sun & Reis, 1992) appropriate control measurements to ethanol application were performed. The volume of ethanol administered (0.03, 0.06 and 0.2 ml) was identical to that required to dissolve the various doses of DPCPX. In this group of animals (n=7), cumulative doses of ethanol were administered intravenously and its effect on the blood pressure and heart rate responses evoked on HDA stimulation examined using an identical protocol to that described for DPCPX.

Intravenous bolus injection of DPCPX, ethanol and 8-SPT

In a third group of animals (n=12), the effect of a single intravenous bolus dose of DPCPX (3 mg kg'% dissolved in 0.25 ml ethanol) and ethanol (0.25 ml) on the defence response was assessed. Control responses to HDA stimulation were performed prior to drug administration. Ethanol was injected slowly into the femoral vein over a period of 5 min and the cannula flushed with an equivalent volume of saline. A 5 min period was allowed following drug administration before a subsequent stimulation of the HDA was performed. The effect of DPCPX on the defence response was then examined in the same animals using an identical protocol. In 3 of these animals femoral blood flow was measured as described above.

In the fourth group of animals (n=6), the effect of 8-SPT was assessed for its effects on the HDA stimulation using an equivalent protocol to described above. Thus, a single intravenous bolus dose of 8-SPT (20 mg kg'% dissolved in 0.4M NaOH and distilled water, final volume 0.20 ml), was injected slowly into the femoral vein 5 min prior to a subsequent stimulation of the HDA.

Histology

At the end of each experiment the stimulation sites within the hypothalamus were lesioned electrolytically and examined histologically (see Section 2.2). Statistical analysis was only undertaken after histological data had been obtained.