We have recently adopted adenoviral vectors to express specific proteins confined to restricted areas of the brainstem to understand nervous control of cardiorespiratory function (Kasparov & Paton, 2000; Paton et al. 2001). Virally mediated gene transfer can be used to enhance the levels of expression of a transmitter/modulator as well as antagonise intracellular pathways through the expression of dominant negative proteins. This approach allows (i) gene manipulation confined to a specific central nervous region of interest, (ii) assessment of the chronic effects of gene expression, (iii) avoidance of problems relating to non-selectivity of drugs or their absence, (iv) control data to be acquired prior to gene intervention and (v) limited time for compensation. All told, viral vectors provide an alternative to transgenic animals.
Normal levels of blood pressure are regulated by arterial baroreceptors that project to neurones residing in the nucleus of the solitary tract or NTS. This structure is essential for cardiorespiratory afferent integration. We have applied adenoviral-mediated gene transfer to the NTS. Specifically we have assessed the mechanisms by which angiotensin II (ANGII) acting within the NTS depresses the baroreceptor reflex, which ultimately allows arterial pressure to rise. Indeed, in hypertension, the gain of the baroreceptor reflex is reduced but blockade of central ANGII type 1 (AT1) receptors helps restore arterial pressure in hypertensive rats (e.g. Gyurko et al. 1993; Phillips et al. 1977).
We found that exogenous ANGII in the NTS depressed the baroreceptor reflex-mediated bradycardia: ANGII suppressed both the reflex activation of cardiac vagal activity and withdrawal of inferior cardiac sympathetic nerve activity during hypertension (Boscan et al. 2001). The depressant effect of ANGII in the NTS on the baroreceptor reflex was mediated via release of nitric oxide (NO). In the absence of selective antagonists for the various isoforms of nitric oxide synthase (NOS), we employed an adenovirus (Ad-TeNOS; see Kantor et al. 1996) to express a dominant negative form of endothelial (e) NOS in the NTS. In these transfected rats, disabling eNOS prevented the depressant effect of ANGII on the baroreceptor reflex (Paton et al. 2001). In addition, in na•ve rats, various NO donors all depressed the reflex. Further, using conventional pharmacological tools we found that ANGII-mediated eNOS activation was calciumÐcalmodulin dependent and occurred through the activation of phospholipase C (PLC): the ANGII-mediated inhibition of the baroreceptor reflex bradycardia was abolished in the presence of a PLC inhibitor U73122 and a calmodulin antagonist Ð W7. The coupling of AT1 receptors to PLC is likely to be mediated by Gq protein. Thus using a different adenoviral vector that expressed a dominant negative form of the α-subunit of the Gq protein, Ad.GaqDN, we blocked Gq-mediated signalling in the NTS. In these transfected animals, exogenous ANGII in the NTS only marginally reduced baroreceptor reflex gain, suggesting that Gq protein is involved in the ANGII-mediated attenuation of the baroreceptor reflex.
Our next quest was to determine the cellular compartment harbouring eNOS. Scanning confocal microscopy revealed that ANGII (200 nM) increased the intracellular calcium concentration of NTS endothelial cells but not neurones. Because intracellular calcium elevation is prerequisite for the ANGII-induced inhibition of the baroreceptor reflex, these data indicate that the endothelium is the primary source of ANGII-triggered release of NO. Moreover, we demonstrate that inhibition of soluble guanylyl cyclase blocked the effect of ANGII. It is therefore likely that NO enhances the efficacy of inhibitory synaptic transmission (GABA) in the NTS, as revealed in earlier studies (Paton & Kasparov, 2000).
Because arterial hypertension is a chronic disease, the long-term effects of gene manipulation in the NTS should be considered also. Using radio telemetry, we measured cardiovascular variables in conscious, freely moving rats in which the NTS was transfected with Ad-TeNOS to disable eNOS. After 2 weeks the baroreceptor reflex gain was enhanced significantly. These data reveal that endogenous eNOS activity within the NTS plays a major role in setting the sensitivity of the baroreceptor reflex and support our previous findings in acute experiments (see above).
We propose a hypothesis of vascular-neuronal signalling in the NTS whereby circulating ANGII releases NO from endothelial cells of the blood vessels that diffuses into the NTS to potentiate the release of GABA, which depresses neurones mediating the baroreceptor reflex. Further, our data demonstrate unequivocally that endogenous eNOS activity in the NTS is a major factor determining baroreceptor reflex function in conscious rats. Whether over-activity of eNOS in the NTS, due to heightened levels of ANGII, results in hypertension awaits investigation.
We thank Dr Erin Schuman and Dr Norman Davidson (California Institute of Technology, Pasadena, CA) for the kind gift of Ad-TeNOS. Ad.GaqDN was a generous donation from Dr David Cook (University of Sydney, Sydney, Australia). The British Heart Foundation funded research (BS 93003 and PG/99055).