The Brain Oxytocin Receptor(s?)

Frontiers in Neuroendocrinology 20, 146–156 (1999) Article ID frne.1999.0178, available online at http://www.idealibrary.com on

COMMENTARY The Brain Oxytocin Receptor(s?) Joseph G. Verbalis Georgetown University School of Medicine, Washington, DC

Remarkable progress has been made over the past decade in understanding the structure and function of many different peptide receptors. Certainly among these have been the receptors for the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT). The picture for the AVP receptors seems reasonably clear, with three major subtypes that have been characterized, of which only the V1 subtype has been localized in the brain parenchyma, while the V2 subtype is found in the collecting tubules of the kidney and the V3 subtype predominantly in the anterior pituitary. While the uterine OT receptor has also been well characterized, knowledge of its functions has lagged somewhat behind that of the AVP receptors. This brief commentary will focus on the OT receptor and specifically on brain OT receptors. I will not attempt to recapitulate the excellent reviews which have been published on neurohypophyseal peptides and their receptors over the past 5 years (7, 8, 24, 27, 75), but rather I will focus on the interesting and important issues which remain to be resolved about the structure, localization, and function of OT receptor(s) in the brain. As an inducement for all neuroendocrinologists to continue reading, it would be decidedly unusual if many of these questions did not bear relevance to understanding analogous aspects of other peptidergic receptors in the brain as well.

STRUCTURE OF OT RECEPTORS

To date only a single OT receptor (OTR) has been cloned from any species. OTR structures have been identified for the human (28), pig (15), rat (52), sheep (50), cow (9), mouse (29), and rhesus monkey (53). All of the cloned receptors are classic G-protein-coupled receptors with seven transmembrane-spanning domains and are highly homologous across species. Because of the high density of

Correspondence and reprint requests may be addressed to the author at 232 Building D, Georgetown University Medical Center, 4000 Reservoir Road NW, Washington, DC 20007. Fax: 202-687-2040. 0091-3022/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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OTRs in the uterus, particularly during pregnancy, most of the identified receptors have been cloned using probes employing sequence information obtained from human myometrial OTR cDNA. However, subsequent studies utilizing reverse transcription polymerase chain reaction methods have identified the gene for the uterine OTR in most all organs previously reported to manifest OT binding, including the mammary gland, pituitary, brain, kidney, thymus, ovary, testes (10, 75), and heart (17), strongly suggesting that the same OTR resides in all these tissues. Furthermore, OTR clones obtained from nonuterine cell lines (e.g., pig kidney LLC-PK1 cells (15) and rat pancreatic RINm5F cells (25)) indicate high degrees of homology with the uterine OTR. Nonetheless, several studies have suggested the presence of different OTR subtypes in some tissues. First, in rats, uterus OTR antagonists block OTinduced myometrial contractile activity but surprisingly cause agonistic effects on endometrial prostaglandin release (11), an action previously attributed to OT stimulation of endometrial OTRs; additional studies showed that, similar to OT, the OT agonist [Thr4,Gly7]OT stimulated myometrial contractions but, unlike OT, it failed to stimulate endometrial prostaglandin release (12). If both of these effects are, in fact, being mediated by OTR activation, this strongly suggests separate endometrial and myometrial OTR subtypes. Second, possible subtypes of OTRs have also been suggested in the kidney, where radioiodine ligand-binding studies have shown that the OT-binding sites in the macula densa and the medulla had similar affinities for OT and OT agonists, but markedly different affinities for AVP and AVP V1 receptor agonists (2). Third, although most brain areas which contain OT-binding sites have been shown to express OT mRNA using cDNA probes derived from the uterine OTR (71), in both the hippocampus and the amygdala antibodies raised against sequences in the rat OTR failed to identify OTR immunoreactivity; in the uterus, mammary gland and other areas of the brain, such immunoreactivity was easily detected (1). Consequently, the possibility of a second OT receptor or OT receptor subtype persists, though the bulk of evidence to date would relegate it to a relatively limited distribution compared to the uterine-type OT receptor. Nonetheless, the presence of a second receptor subtype could help explain differential tissue responses to OT antagonists and agonists, as well as possibly some of the differential regional effects of steroid hormones on OT receptor expression discussed below. Some valuable lessons about elusive receptor subtypes should be recalled from the story of the discovery of the various angiotensin receptors. The rat angiotensin AT1a and AT1b receptors have 95% homology at the amino acid level but are derived from separate genes located on two different chromosomes [17 and 2, respectively (32)]. They have subtle differences in their pharmacology and signaling mechanisms, but widely different tissue distributions likely due to differences in their 58 flanking regions (54). Of even greater relevance, the angiotensin AT2 receptor shares ⬍30% homology with the angiotensin AT1 receptors. The AT1 and AT2 receptors have identical affinities for angiotensin, but relatively modest differences in affinities toward other angiotensin analogues in various tissues hinted at the possibility of different angiotensin

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receptor subtypes. However, it was not until various pharmaceutical companies developed different receptor antagonists that it became clear that at least two pharmacologically distinct AT receptors existed. In this case Southern analysis would not have revealed two receptor subtypes because the homology was so low between the AT1 and AT2 receptors; the second receptor (AT2) had to be cloned to conclusively prove its existence (13, 51, 66, 69). Consequently, a second OTR could quite conceivably exist that has low homology to the cloned uterine receptor and so it would not show up on Southern analysis or by PCR, but would potentially explain the varied pharmacological profiles of OTRs in different tissues. A second issue which has resulted in continued uncertainty is the transcriptional control of OTRs. Initial analysis of the 58 flanking regions of the rat (52) and human (18) OTR genes indicated consensus sequences for several promotor elements, including nuclear-factor interleukin-6 (NF-IL6) and the acute-phase response factor (STAT3). Neither gene contained classical estrogen response elements (EREs) although several partial ERE sequences (‘‘half-palindromes’’) were identified. This was more than a little surprising, because the OTR is one of the most responsive genes to estrogen stimulation, with clear increases in both receptor binding by autoradiography and receptor mRNA expression by in situ hybridization in the uterus, the ventromedial hypothalamus (VMH), and the kidney (3, 43, 61, 65). Furthermore, a classical ERE as well as several half-palindromic motifs were identified in the 58 flanking region of the mouse OT receptor gene (29). This dilemma has now been clarified, at least in the rat, by further cloning of the OTR gene which has indicated a classical ERE in a previously unidentified 1260-nucleotide sequence from the 58 upstream flanking region which indeed confers estrogen responsivity in vitro (5). Further studies of the 58 upstream region have indicated other promotor sequences as well, including AP-1, AP-2, AP-3, and AP-4 sites (6). Whether similar sequences are present in the human gene and that of other species has not yet been ascertained, but presumably will be explored in the near future. Given this wealth of new information, what questions still remain to be answered about OTR structure? First, and undoubtedly foremost, is there more than one OTR subtype? And if so, do these represent splice variants of a single gene or the product of a completely separate gene? Second, if there is only a single gene, what accounts for some of the differential tissue responses to OT agonists and antagonists? Might this instead be a reflection of different coupled second messenger systems in different tissues? Third, can the unique steroid responsiveness of the OT gene be entirely accounted for by an ERE in the 58 leader sequence region in all species? And to what degree is the dramatic estrogen responsivity of brain regions such as the VMH accounted for solely by quantitative changes in OT receptor expression?

LOCALIZATION OF OT RECEPTORS

Accurate information about the distribution of OT receptors in the brain has come from receptor autoradiography studies, which have been dramatically

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enhanced by the development of very specific radioiodinated ligands for AVP and OT (26, 60). A convincing proof of the specificity of current radioiodinated AVP and OT receptor ligands is the essentially nonoverlapping nature of AVPand OT-binding sites in the brain (8). Prominent OT receptor binding has been found in the anterior olfactory nucleus, the bed nucleus of the stria terminalis, the ventral nucleus of the amygdala, the hippocampal ventral subiculum, the ventromedial nucleus of the hypothalamus, the dorsal motor nucleus of the vagus nerve, and the substantia gelatinosa and intermediolateral cell columns of the spinal cord in the rat. Confirmation that areas possessing these binding sites indeed express OTRs comes from more recent in situ hybridization studies using cDNA probes from the cloned human and rat OTR gene sequences. Initial studies used probes to the human OTR in rat brain (71) and demonstrated transcripts in essentially all areas previously found to have OT-binding sites by receptor autoradiography with the sole exception of the inferior olive. Several other areas were found to have disproportionately high OTR mRNA levels compared to radioligand-binding sites, including the dorsal vagal motor nucleus of the vagus, the anterior medial preoptic nucleus (AV3V area), and the hypothalamic supraoptic and paraventricular nuclei. The latter areas are of particular interest, since abundant physiological evidence has indicated that centrally released OT can stimulate the firing of magnocellular OT neurons as well as the pituitary release of OT (30, 39, 40, 70). Since these effects appear to be produced by dendritically released OT (31, 34), it is possible that the OTRs are similarly located more distantly on dendrites of OT neurons accounting for the failure of radioligand-binding studies to detect binding sites in the magnocellular regions of these nuclei. Multiple in situ hybridization studies utilizing probes derived from rat OTR cDNA have yielded fairly similar results in terms of localization of the receptor transcripts (3, 4, 48, 72). More interestingly, the latter studies evaluated tissue-specific changes in OTR mRNA levels in response to physiological and pharmacological stimuli. All studies agree that estrogen causes a marked up-regulation of OTR mRNA in the rat VMH. Some of the studies have shown lesser (amygdala, hippocampus, and anterior pituitary) or no (arcuate, caudate putamen) increases in other areas (48), while others have failed to see significant estrogen-induced increases in any areas other than the VMH (3, 72). As might be expected from the identification of NF-IL6 promotor sequences on the 58 leader sequence of the rat OT gene, IL6 significantly potentiated the estrogen-induced increase in OTR mRNA in the VMH (72), demonstrating modulation of a basic estrogen-induced response by a cytokine which is known to be released by the placenta at the onset of parturition (42). The marked differences in estrogen sensitivity in different parts of the brain can likely be attributed to differential expression of estrogen receptors in different tissues. Studies in mice genetically deficient in estrogen receptor ␣ (ER␣) have shown that this receptor is essential for estrogen-induced increases in OTR binding (73). Although ER␤ has been localized to various brain regions including the lateral septum, amygdala, hippocampus, and PVN (58), ER␣ is well known to be heavily expressed in the basal hypothalamus (57). Finally, it is of interest that pregnancy has been found to increase OTR

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mRNA expression in many areas not significantly affected by estrogen administration, including the lateral septum and medial preoptic area (72). This serves to reemphasize the important point that brain OT is involved in more physiological and behavioral functions than simply estrogen-stimulated reproductive behaviors. In this case, these region-specific changes in OT receptor expression during pregnancy likely underlie the induction of maternal behaviors in pregnant rats prior to parturition (46). In view of these significant advances, what questions remain to be answered about OTR localization? First, future studies will be needed to address the colocalization of ER␣ and OTRs on cells in the VMH compared to less estrogenresponsive areas. Techniques for visualizing dual localization of steroid and peptide receptors on neurons are now sufficiently advanced that this question should be answerable in the near future. Second, can other steroids also affect OTR expression? To date, both progesterone (44, 55) and glucocorticoids (33) have been reported to affect OTR expression in the brain. Third, does steroid regulation of OTR expression occur differentially in separate regions of the brain? One intriguing example of this is that both glucocorticoids and stress appear to up-regulate OTR expression in the hippocampus, an area rich in glucocorticoid receptors more so than OTRs (33). Finally, the nagging issue of receptor–peptide mismatch still lurks beneath the surface of today’s seemingly otherwise consistent results. Despite widespread acceptance of the concept of volume transmission to explain peptidergic effects on cells in the absence of demonstrable peptidergic afferent inputs, even this potential mechanism stretches the imagination in areas such as the amygdala, which possesses one of the highest OTR-binding activities in the brain but with very few OT afferents nearby. Examples such as this will continue to raise questions as to whether there are other endogenous ligands that preferentially bind to and activate OTRs in these areas.

FUNCTION OF OT RECEPTORS

Although many questions remain about the structure and localization of OTRs, the most exciting and important area of future study will clearly be the elucidation of the function of OTRs in the brain. Molecular biology and anatomy are essential tools, but only studies of receptor function can help us understand the involvement of brain peptides in physiological and pathophysiological processes. On a cellular level, it will be important to understand the second messenger systems activated by ligand binding to OTRs. Much information is already known about the uterine OTR, which stimulates phosphatidylinositide turnover, activation of PKC, and increases in intracellular calcium (47). Recent studies have suggested that increases in cAMP acting through PKA can antagonize OT-mediated phosphatidylinositide turnover, thus acting as a brake on OT activation (14). However, to date none of the OT-coupled second messenger systems in the brain have been definitively identified. Preliminary results have suggested that OTR second messenger coupling may, in fact, vary in

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different parts of the brain, again raising the question of different receptor subtypes and/or signaling systems in different regions of the brain (37). A second area of great recent interest is the potential for nongenomic effects of steroids on OTRs. Along with earlier suggestions (56), recent studies of the effects of progesterone on uterine OT binding have suggested a nongenomic action of this steroid to directly interfere with OT agonist binding to membranes containing OTRs (16). Although nongenomic effects of steroids have been postulated to affect the function of a variety of cell-surface receptors, including the GABAA, NMDA, and nicotinic acetylcholine receptors (35, 62, 68), this purported effect on OTRs is the first time such effects have been demonstrated to influence G-protein-coupled receptors. If confirmed, these results could help explain the ability of progesterone to maintain uterine quiescence by dampening uterine sensitivity to circulating OT levels during pregnancy. Similarly, antagonistic effects of progesterone on central OT functions in the absence of changes in OTR expression would be easier to understand. Beyond defining cellular actions of OT on brain OTRs, the larger challenge of understanding the effects of OT on neuronal systems involved with physiological and behavioral functions remains. As already discussed, the unique estrogen sensitivity of the OTR confers the certainty of a key role in reproductive behaviors. Indeed, multiple lines of evidence support the involvement of brain OT, acting on cerebral OTRs, in both reproductive and maternal behaviors (23, 36, 45). Perhaps the most important of these from the point of view of brain OTRs have been studies of administration of antisense oligonucleotides directed against the OTR, which demonstrated that such injections into the VMH were able to block specific reproductive behaviors such as lordosis in the female rat (38). Coupled with studies demonstrating up-regulation of OTR expression with estrogen administration and pregnancy (48, 72), there can be little doubt about the important and unique role the OTR plays in such functions. Indeed, the OTR appears to be the quintessential example of a gonadal steroidregulated G-protein-coupled receptor in the brain; there are no close competitors on the horizon. Very likely closely related to its influence on maternal behaviors is the involvement of brain oxytocinergic systems on a variety of social behaviors. The remarkable species-specific variations in OTR distribution in different species of the prairie vole which have been found to correlate closely with their proclivity to social interactions versus aggressiveness (21, 22) raises the obvious question of whether a similar diversity of OTR distributions, both across and within species, and perhaps even between sexes, can account for often profound differences in affiliative versus aggressive behaviors (19,20). Recent studies of OT knockout mice have supported this hypothesis (74). However, if this is true, then the reported paucity of OTRs in the primate brain may not bode well for the future of conflict resolution in our society (59). Despite the obvious importance of brain OTRs for reproductive and maternal behaviors, it should also be remembered that no peptidergic/receptor systems are monolithic; the varied functions of brain peptides have diverged in response to evolutionary pressures in ways that are both amazingly varied and unpredict-

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able. A particularly apt example of this is that one of the best-characterized central functions of AVP, acting on brain V1a receptors, is antipyresis (67). Who would have predicted that a peptide predominantly involved peripherally with renal water excretion and vasoconstriction would play a major thermoregulatory role via its central actions? Similarly, it is important to remember that brain OT has been implicated in multiple actions seemingly unrelated to its effects on reproductive and maternal behavior, including social behaviors, memory, gastric function, and ingestive behaviors (49). However, it may yet turn out that some of these effects indeed serve a common integrative purpose which we have simply been too narrow minded to fully appreciate. The aforementioned relation between maternal and affiliative behaviors is an obvious example of this. Another may be OT inhibition of food intake. The seeming uncharacteristic nature of this response has cast doubt on its veracity. Since this effect appears to occur only when OT secretion is stimulated, it is logical to consider when this occurs. In rats, OT secretion is stimulated in response to suckling, parturition, and hyperosmolality. In suckled rats, a maternal interruption of suckling activity to forage for food would clearly be detrimental to pup survival and therefore to species survival as well. A similar argument could be made for interruption of reproductive behavior in response to hunger. The absence of such behaviors from mammalian species has generally been attributed to sexual and maternal drives overriding more vegetative behaviors such as feeding. However, it seems just as likely that complementary controls of food intake act to eliminate or reduce this as a competing behavior during the important acts of species procreation and nurturing. Finally, reduced food intake during states of hyperosmolality would prevent exacerbation of hyperosmolality due to the solute load inherent in ingested food, which would explain why OT appears to be inhibitory to both salt and food intake (63, 64). When viewed in this context, perhaps brain OT and OTR functions will eventually provide an example of multiple, seemingly diverse brain effects which nonetheless subserve a common integrative purpose, as has the effects of brain CRH to integrate the complex organismal response to stress. In keeping with the format of the previous sections, it is appropriate to close by asking what questions still remain to be answered about brain OTR function? Quite simply, in my opinion everything still remains to be discovered or at least better understood, from deciphering second messenger systems, to differentiation of genomic versus nongenomic effects of steroids, to unraveling the complex physiological and behavioral effects mediated or modulated by brain OT and OTRs. This is indeed a field ripe for major advances in the very near future, as the existing powerful tools of molecular biology, anatomy, physiology, and behavior are brought to bear on this evolutionarily ancient yet very current and relevant neuroendocrine system. In particular, the careful application of appropriate physiological and behavioral studies to animals with OTR gene knockouts to clarify and expand initial results obtained with OT knockout mice (41), as well as novel techniques such as developing OTR transgenic mice with altered promotor sequences that change OTR distributions in the brain (74), will likely yield new insights into the function of OTRs in

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the brain and elsewhere. Consequently, I feel quite comfortable predicting that a follow-up commentary several years from now will summarize the answers to all of these questions and to others not yet even imagined.

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72. 73. 74.

75.

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