Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single-channel behaviour.

1. In order to establish the subunit composition of neuronal nicotinic receptors in rat superior cervical ganglia (SCG), their single-channel properties were compared with those of recombinant receptors expressed in Xenopus oocytes, using outside-out excised patch recording. 2. The mean main conductance of SCG channels from adult and 1-day-old rats was 34.8 and 36.6 pS, respectively. Less frequent openings to lower conductances occurred both as isolated bursts and as events connected to the main level by direct transitions. There was considerable interpatch variability in the values of the lower conductances. 3. Nicotinic receptors from oocytes expressing alpha3beta4 and alpha4beta4 subunits had chord conductances lower than that of SCG neurones (22 pS for alpha3beta4 and 29 pS for alpha4beta4). 4. Prolonged recording from both native and recombinant channels was precluded by 'run-down', i.e. channel activity could be elicited for only a few minutes after excision. Nevertheless, SCG channel openings were clearly seen to occur as short bursts (slowest component, 38 ms), whereas recombinant channels opened in very prolonged bursts of activity, the major component being the slowest (480 ms). 5. Addition of the alpha5 subunit to the alpha3beta4 pair produced channels with a higher conductance than those observed after injection of the pair alone (24.9 vs. 22 pS), suggesting incorporation of alpha5 into the channel. Addition of the beta2 subunit did not change alpha3beta4 single-channel properties. In one out of fourteen alpha3alpha5beta4 patches, both ganglion-like, high conductance, short burst openings and recombinant-type, low conductance, slow burst openings were observed. 6. Channels produced by expression in Xenopus oocytes of neuronal nicotinic subunits present in rat SCG as a rule differ from native ganglion receptors in single-channel conductance and gross kinetics. While it is possible that an essential nicotinic subunit remains to be cloned, it is perhaps more likely that oocytes either cannot assemble neuronal nicotinic subunits efficiently into channels with the correct composition and stoichiometry, or that they produce post-translational channel modifications which differ from those of mammalian neurones.

The influence of neurokinins and calcitonin gene-related peptide on cerebral blood flow in anaesthetized guinea-pigs.

The effects of systemically-administered human alpha calcitonin gene-related peptide (h.alpha CGRP), substance P and the selective neurokinin receptor agonists, GR73632 (NK1) and GR64349 (NK2) on cerebral blood flow (CBF) were studied in anaesthetized guinea-pigs using a laser-Doppler flowmeter. h.alpha CGRP (0.1 and 0.3 nmol/kg), substance P (0.03-1.0 nmol/kg), GR73632 (0.03-0.3 nmol/kg) and GR64349 (0.3 nmol/kg) each, following intra-carotid artery injection, reduced transiently (< 5 min) blood pressure and CBF. GR73632 (0.1 and 0.3 nmol/kg) and GR64349 (0.3 nmol/kg), but not h.alpha CGRP (0.01-0.3 nmol/kg) or substance P(0.01-1.0 nmol/kg), then produced a more prolonged increase in CBF, the peak effect occurring 10-15 min after injection. It is likely that this increase in CBF was due to their bronchoconstrictor activity, rather than a direct effect on the cerebrovasculature; arterial PaCO2 levels were increased and PaO2 decreased by both compounds. Following pretreatment with urea (5 M) to disrupt the blood brain barrier, h.alpha CGRP (0.1 nmol/kg) produced a significant increase in CBF (13 +/- 4%), implying that access to its receptors on the cerebrovascular smooth muscle had been achieved. Substance P (0.1 nmol/kg) remained inactive. The study has demonstrated that compounds acting on neuropeptide receptors have little direct influence on CBF following systemic administration. CGRP requires access to its receptors on the cerebrovascular smooth muscle, while selective NK1 and NK2 receptor agonists increase CBF, probably indirectly via their bronchoconstrictor activity. The lack of effect of substance P may be due to its rapid breakdown by peptidases, a property not shared by the selective neurokinin agonists.