Synergies at the synapse.

Genetically engineered mice continue to provide insights into the details of physiology beyond what investigators might have originally expected. Despite the formal possibility that gene ‘knockout' or other targeted gene manipulations might lead to perturbed development and highly confounded interpretations of changes in physiology measured in adult mice, these limitations are not routinely faced following perturbation of genes encoding G protein-coupled receptor (GPCR) systems. In fact, it has been reasonably straightforward to interpret the findings in animals where mutation or deletion of genes encoding GPCRs have been undertaken. Perhaps this is because GPCRs typically effect modulatory roles–not regulating on–off switches (except perhaps in sensory systems), but changing the signal frequency or intensity of ongoing cellular activity. One example where gene targeting has had a profound impact is on the understanding of the sympathetic nervous system, in general, and for the roles of α2-adrenoceptor (AR) subtypes, in particular. There are three subtypes of α2-ARs (dubbed α2A-, α2B-, and α2C-AR). These receptor subtypes are encoded by three distinct, intronless genes that exist on each of three chromosomes in human beings as well as in mice. Mutation of the α2A-AR to D79N α2A-AR or creation of null alleles for the α2A-, α2B-, and α2C-AR subtypes has revealed the roles of these subtypes in various physiological and behavioral paradigms (Link et al., 1996; MacMillan et al., 1996; Lakhlani et al., 1997; Rohrer & Kobilka, 1998; Altman et al., 1999). The α2A-AR appears to be the subtype that plays the principal role in response to α2-agonists for the suppression of blood pressure, attenuation of pain perception, analgesia, anesthetic sparing, and suppression of neurotransmitter release (MacMillan et al., 1996; Lakhlani et al., 1997; Altman et al., 1999). In contrast, it is the α2B-AR subtype that elicits the pressor effect of α2-agonists and, as well, plays a role in salt-induced hypertension (Link et al., 1996; Rohrer & Kobilka, 1998). The α2C-AR subtype, as will be discussed below, also contributes to the suppression of neurotransmitter release and, in addition, has effects on behavioral systems, such as altering the swimming behavior of mice – an experimental paradigm for testing the efficacy of antidepressant agents (Rohrer & Kobilka, 1998). As in any other area of scientific exploration, the more rigorous the biological assessment, the greater the detail and understanding achieved by the studies. An elegant example of this is in the studies of Hein et al. (1999), who demonstrate in α2A/C-AR ‘double knockout' mice that both the α2A- and α2C-AR subtypes contribute to the regulation of neurotransmitter release and do so in a frequency-dependent fashion. Thus, it is the α2A-AR that suppresses norepinephrine in response to high-frequency and high-intensity electrical stimulation, whereas the α2C-AR attenuates norepinephrine release at lower stimuli and frequency intervals. In fact, the role of the α2C-AR is appreciated only when both the α2A- and α2C-AR subtypes have been eliminated (Hein et al., 1999). The reason that the loss of both receptor subtypes must occur for the α2C-AR role in the suppression of neurotransmitter release to be detected is not clear. One wonders if hetero-oligomers of α2-AR subtypes might occur in cells where both α2A- and α2C-AR subtypes are expressed, and have unexpected influence in α2C-AR function, by analogy with hetero-oligomers of opioid receptor subtypes (Jordan & Devi, 1999). The paper by Trendelenburg et al. (2003) in this issue of the British Journal of Pharmacology represents a continuation of rigorous analysis of the interplay of Gi/Go-coupled receptors (such as the α2-AR autoreceptor) and Gq-coupled receptors on tritium overflow from tissues pre-equilibrated with 3H-noradrenaline. The present findings follow up on earlier observations that angiotensin and bradykinin enhance tritium overflow from mouse atria by extending these studies to vas deferentia stimulated under various degrees of α2-AR inhibition (Cox et al., 2000). These studies were devised to explore in more detail the crosstalk between angiotensin (AT1) and bradykinin (B2) receptors, where it is shown that the Gi/Go system must be engaged by the α2-AR in order for the Gq-enhanced stimulation of tritium overflow to be detected. Thus, incubation with antagonists at the α2-AR (such as phentolamine and rauwolscine) or elimination of α2-AR autoreceptor function by gene deletion, attenuates and eliminates, respectively, enhancement of tritium overflow by AT1 and B2 receptors. This interplay between Gi/Go- and Gq-coupled receptors is not limited to α2-ARs, however. In the presence of phentolamine or the absence of α2-AR gene expression, the effects of AT1 or B2 receptor agonists to enhance tritium overflow can be mimicked by engaging Gi/Go-coupled neuropeptide Y, cannabanoid, or μ-opioid receptors. The interesting and obvious question arises as to what might represent the molecular bases for the interesting physiological findings observed. The findings are reminiscent of studies summarized in 1993 by Bourne & Nicoll (1993), concerning coincident signaling in neurotransmitter systems. Bourne & Nicoll (1993) postulated that convergence on differing isoforms of adenylyl cyclase could explain synergism of Gs- and Gi-coupled pathways, or, alternatively, Ca2+- and Gs-coupled pathways, which are functionally antagonistic of one another when activated individually or in nontemporally linked experimental paradigms, but are synergistic upon paired or coincident activation. These authors proposed that the varying isoforms of cyclase, with different sensitivities to βγ subunits, Ca2+ or coincident regulation by Gs and βγ subunits, represented the molecular machines that integrated these disparate neuronal inputs. It is unlikely that the enzyme adenylyl cyclase represents the molecular integrating machine in the physiological settings under study, particularly since Starke and colleagues have previously shown that Gs-mediated β-adrenoceptor enhancement of tritium overflow is independent of engagement of Gi/Go-coupled receptor systems. Nonetheless, it is possible that a similar molecular integration occurs at the level of the receptor-operated K+ channels, voltage-gated Ca2+ channels, or synaptic vesicle fusion complexes intimately involved in frequency-modulated neurotransmission in the sympathetic nervous system. The interplay enhancement of tritium overflow by AT1 and B2 receptors was observed to occur with engagement of any number of Gi/Go-coupled receptors tested in these experiments (NPY, μ-opioid receptor, cannabanoid receptor, and α2-AR). Interestingly, this crosstalk required the α2C-AR, not the α2A-AR, subtype. The observation that it is the α2C-AR subtype that plays the predominant role in determining the extent of tritium overflow in response to Gq-coupled agonists suggests that Gi-coupled changes in membrane potential, particularly via response to low-frequency stimulation (a property of the α2C-AR subtype, in particular; Hein et al., 1999), might account for this response. The findings of Trendelenburg et al. implicate a crosstalk between Gq- and Gi/Go-coupled pathways, which encourages further exploration to reveal its molecular basis – be it receptor oligomers, microcompartments, scaffolds, or integrative ‘molecular machines' composed of one or several molecular players. The findings do re-emphasize, however, the intricate molecular awareness of the synaptic junction of just which neurotransmitter receptors, and their effector pathways, are engaged, exquisitely synthesizing biochemical and electrical inputs to achieve appropriate trans-synaptic communication.