Diverse roles of G-protein coupled receptors in the regulation of neurohypophyseal hormone secretion.

The magnocellular neurones in the supraoptic nucleus project to the neural lobe and release vasopressin and oxytocin into the peripheral circulation, where they act on the kidney to promote fluid retention or stimulate smooth muscles in the vasculature, uterus and mammary glands to support blood pressure, promote parturition or induce milk let-down, respectively. Hormone release is regulated by complex afferent pathways carrying information about plasma osmolality, blood pressure and volume, cervical stretch, and suckling. These afferent pathways utilise a broad array of neurotransmitters and peptides that activate both ligand-gated ion channels and G-protein coupled receptors (GPCRs). The ligand-gated ion channels induce rapid changes in membrane potential resulting in the generation of action potentials, initiation of exocytosis and the release of hormone into the periphery. By contrast, the GPCRs activate a host of diverse signalling cascades that modulate action potential firing and regulate other cellular functions required to support hormone release (e.g. hormone synthesis, processing, packaging and trafficking). The diversity of these actions is critical for integration of the distinct regulatory signals into a response appropriate for maintaining homeostasis. This review describes several diverse roles of GPCRs in magnocellular neurones, focusing primarily on adrenergic, purinergic and peptidergic (neurokinin and angiotensin) receptors.

Characterization of nuclear neurokinin 3 receptor expression in rat brain.

Ligand-induced translocation of the G-protein-coupled receptor, neurokinin 3 (NK3-R), to the nucleus of hypothalamic neurons was reported using antibodies (ABs) raised against the C-terminal region of NK3-R. The current work was undertaken to substantiate the ability of NK3-R to enter the nucleus and identify which portion of the NK3-R molecule enters the nucleus. ABs directed at epitopes in the N-terminal and second extracellular loop of the rat NK3-R molecule were used to evaluate western blots of whole tissue homogenates and nuclear fractions from multiple brain areas. Specificity of the protein bands recognized by these ABs was demonstrated using Chinese hamster ovary (CHO) cells transfected with rat or human NK3-R. Both ABs prominently recognized a diffuse protein band of ∼56-65 kDa (56 kDa=predicted size) and distinct ∼70-kDa and 95-kDa proteins in homogenates of multiple brain areas. The ∼95-kDa protein recognized by the extracellular loop AB was enriched in nuclear fractions. Recognition of these proteins by ABs directed at different regions of the NK3-R supports their identification as NK3-R. The size differences reflect variable glycosylation and possibly linkage to different cytosolic and nuclear proteins. Recognition of protein bands by both ABs in nuclear fractions is consistent with the full-length NK3-R entering the nucleus. Hypotension increased the density of the ∼95-kDa band in nuclear fractions from the supraoptic nucleus indicating activity-induced nuclear translocation. Since NK3-R is widely distributed in the CNS, the presence of NK3-R in nuclei from multiple brain regions suggests that it may broadly influence CNS gene expression in a ligand-dependent manner.

Site of ATP and phenylephrine synergistic stimulation of vasopressin release from the hypothalamo-neurohypophyseal system.

ATP and norepinephrine are neurotransmitters carrying cardiovascular information to vasopressin (AVP) neurones. As shown previously, exposure of hypothalamo-neurohypophyseal system explants to ATP and phenylephrine (PE) (alpha(1)-adrenergic agonist) causes a significantly larger increase in AVP release than with either agent alone and converts the response from a transient to a sustained stimulation of AVP release. Potential mechanisms for this synergism include presynaptic stimulation of excitatory afferent input (i.e. glutamate release), postsynaptic activation of receptors on AVP neurones, modulation of stimulus-secretion coupling in the neural lobe and/or involvement of glial/neuronal interactions. The response to ATP + PE (100 microM each) was not altered in the presence of either a cocktail of ionotropic glutamate receptor antagonists (CNQX + AP5) or a nonselective metabotropic glutamate receptor antagonist [(RS)-alpha-methyl-4-carboxyphenylglycine]. Thus, it is not dependent on activation of glutamate receptors. Treatment with tetrodotoxin (3 microM) eliminated the response to ATP + PE. Because this could reflect blockade of action potentials propagated from the AVP perikarya to the nerve terminals in the neural lobe or action potentials generated in the neural lobe directly, synergism in the neural lobe was addressed by perifusing isolated neural lobes with ATP and PE alone or together. Synergistic stimulation of AVP release by ATP + PE was not observed in isolated, perifused neural lobes. Thus, the synergistic effect persists in the absence of glutamate transmission, is not due to synergistic actions of P(2) and alpha(1)-adrenergic receptors occurring at the level of the neural lobe and requires action potentials in either the hypothalamus or neural lobe.

Depletion of oestrogen receptor-beta expression in magnocellular arginine vasopressin neurones by hypovolaemia and dehydration.

Oestrogen receptor (ER)-beta expression correlates inversely with osmotic control of arginine vasopressin (AVP) release such that cellular dehydration induced by 72 h of 2% saline consumption depletes ER-beta in the magnocellular AVP neurones in the supraoptic (SON) and paraventricular nuclei (PVN). The current studies were performed to determine whether other pathways that stimulate AVP release, such as hypovolaemia, also regulate ER-beta expression in these nuclei, and to evaluate the time course of the change in ER-beta expression during water deprivation and subsequent rehydration. ER-beta expression was evaluated immunocytochemically. In rats made hypovolaemic with a subcutaneous injection of 40% polyethylene glycol (PEG), a significant depletion of ER-beta in both SON and magnocellular PVN (P

Oestrogen receptor beta: role in neurohypophyseal neurones.

The robust expression of oestrogen receptor beta (ER-beta) in magnocellular vasopressin neurones has focused attention on the role of this receptor and the gonadal steroids in the regulation of vasopressin secretion. Although the effects of gonadal steroids on vasopressin secretion have been the subject of many studies, there is no consensus in the literature as to their role. Possible reasons for the diverse findings are discussed, including diversity in the types, site and level of expression of steroid receptors across species, gender and physiological conditions. The physiological regulation of expression is of particular interest because ER-beta mRNA expression in vasopressin neurones is inversely correlated to the osmotic state of the animal. Chronic hyperosmolality inhibits ER-beta mRNA expression in magnocellular vasopressin neurones, while chronic hypo-osmolality enhances expression. This is consistent with an inhibitory role for ER-beta because hyperosmolality is a potent stimulus for vasopressin secretion, whereas vasopressin secretion is maximally inhibited by chronic hypo-osmolality. An inhibitory role is also indicated by in vitro experiments demonstrating inhibition of osmotically stimulated vasopressin secretion by oestrogen and testosterone, and ER-beta mediated inhibition of NMDA-stimulated vasopressin secretion. The challenge remains to elucidate the mechanism of this inhibition, and to understand its significance for maintenance of whole-body fluid and electrolyte homeostasis.

Neurotransmitter/neuropeptide interactions in the regulation of neurohypophyseal hormone release.

Regulation of neurohypophyseal hormone release reflects the convergence of a large number of afferent pathways on the vasopressin (VP)- and oxytocin-producing neurons. These pathways utilize a broad range of neurotransmitters and neuropeptides. In this review, the mechanisms by which this information is coordinated into appropriate physiological responses is discussed with a focus on the responses to agents that are coreleased from A1 catecholamine nerve terminals in the supraoptic nucleus. The A1 pathway transmits hemodynamic information to the vasopressin neurons by releasing several neuroactive agents including ATP, norepinephrine, neuropeptide Y, and substance P. These substances stimulate VP release from explants of the hypothalamo-neurohypophyseal system and certain combinations of these agents elicit potent but selective synergism. Evaluation of the signal cascades elicited by these agents provides insights into mechanisms underlying these synergistic interactions and suggests mechanisms responsible for coordinated responses of the VP neurons to activation of a range of ion-gated ion channel and G-protein-coupled receptors.

Effect of forskolin and exogenously administered oxytocin mRNA on oxytocin release by dispersed hypothalamic cultures.

Differential vasopressin (VP) gene expression and oxytocin (OT) gene expression were observed in hypothalamic cultures derived from 14-day-old rat fetuses, with VP but not OT being induced by treatment with forskolin and 3-isobutyl-1-methylxanthine. These cultures were used to demonstrate that exogenous VP mRNA could be taken up and translated into releasable VP. In the current studies a similar culture preparation was used to test the hypothesis that, due to the similarity in the mRNA and prohormone structures of VP and OT, the VP-expressing neurons in the cultures would be capable of utilizing exogenous OT mRNA for synthesis of releasable OT. Although OT release was increased by the administration of exogenous OT mRNA, endogenous OT gene expression was also observed. To determine what had induced OT gene expression in the current cultures, the undefined components of the culture preparation, e.g., the glial feeder layer and the serum component of the culture medium, were evaluated. Restraining growth of the glial carpet with cytosine-arabinoside did not alter OT gene expression. Use of a defined medium supplemented with B-27 induced optimal OT gene expression. From this, it is possible to conclude that the components included in B-27 are sufficient for OT gene expression.Factors included in earlier lots of sera may have been responsible for suppression of OT gene expression. Cultures maintained in serum-free, B-27-supplemented medium may provide a useful model system for studying OT gene regulation.

Role of metabotropic glutamate receptors in vasopressin and oxytocin release.

The effect of metabotropic glutamate receptor (mGluR) activation on vasopressin (VP) and oxytocin (OT) release was evaluated using explants of the hypothalamoneurohypophysial system. (+/-)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), an agonist at groups I and II mGluRs, increased VP and OT release in a concentration-dependent manner. A role for group I mGluRs in VP and OT release was demonstrated by the ability of a group I-specific mGluR antagonist, 1-aminoindan-1,5-idicarboxylic acid (AIDA), to block the effect of t-ACPD and the ability of a group I-specific agonist, (R,S)-3,5-dihydroxyphenylglycine, to significantly increase both VP (P = 0.0029) and OT (P = 0.0032) release. However, AIDA did not alter VP or OT release induced by a ramp increase in osmolality of the perifusion medium. The role of group III mGluRs was examined using L(+)-2-amino-4-phosphonobutyric acid (L-AP4), an agonist of these receptors. L-AP4 did not change basal release of VP or OT and did not prevent osmotically stimulated hormone release. Thus mGluR activation stimulates VP and OT release, but it is not required for osmotic stimulation of hormone release.

Role of non-NMDA receptors in vasopressin and oxytocin release from rat hypothalamo-neurohypophysial explants.

Glutamate is recognized as a prominent excitatory transmitter in the supraoptic nucleus (SON) and is involved in transmission of osmoregulatory information from the osmoreceptors to the vasopressin (VP) and oxytocin (OT) neurons. Explants of the hypothalamo-neurohypophysial system were utilized to characterize the roles of the non-N-methyl-D-aspartate (NMDA) glutamate receptor subtypes (non-NMDA-Rs), kainic acid receptors (KA-Rs), and aminopropionic acid receptors (AMPA-Rs) and to evaluate the interdependence of NMDA-Rs and non-NMDA-Rs in eliciting hormone release. Although both KA and AMPA increased hormone release, a specific agonist of the KA-Rs, SYM-2081, was not effective. This combined with the finding that cyclothiazide, an agent that inhibits the desensitization of AMPA-Rs, increased the VP response to both KA and AMPA indicates that the increase in hormone release induced by the non-NMDA agonists is mediated via AMPA-Rs, rather than KA-Rs. Inhibition of osmotically stimulated VP and OT release by a specific AMPA-R antagonist indicated that AMPA-Rs are essential for mediating osmotically stimulated hormone release. NMDA-stimulated VP but not OT release was prevented by blockade of non-NMDA-Rs, but AMPA-stimulated VP/OT release was not prevented by NMDA-R blockade.

cAMP stimulation of vasopressin and oxytocin release and regulation of vasopressin mRNA stability: role of auto-facilitation.

The effects of cycloheximide and actinomycin on 8-bromo-cAMP (8-Br-cAMP) stimulated vasopressin and oxytocin release from the posterior pituitary and vasopressin mRNA content of the supraoptic nucleus were studied with perifused explants of the hypothalamo-neurohypophyseal system. 8-Br-cAMP stimulated vasopressin and oxytocin release from the explant for up to 6 h. Inhibition of protein synthesis by cycloheximide completely suppressed the response to 8-Br-cAMP. When gene transcription was inhibited by actinomycin, vasopressin release was stimulated by 8-Br-cAMP for approximately 2 h, but the response was not sustained. Vasopressin mRNA content was not changed by 8-Br-cAMP in the absence or presence of cycloheximide, but it was significantly decreased by simultaneous exposure to 8-Br-cAMP and actinomycin. Actinomycin alone did not change vasopressin mRNA content. Since other studies have demonstrated that cAMP stimulates vasopressin gene transcription, and since vasopressin mRNA content reflects the balance between gene transcription and mRNA degradation, the effect of actinomycin and 8-Br-cAMP on vasopressin mRNA content suggests that 8-Br-cAMP also decreased vasopressin mRNA stability and thereby induced a rapid turnover of vasopressin mRNA. The effects of cycloheximide and actinomycin on vasopressin and oxytocin release suggest that ongoing protein synthesis is required for stimulation of hormone release. Since the posterior pituitary hormone stores are not depleted with a stimulus for release that is even more potent than cAMP, it is possible that cycloheximide and actinomycin depleted smaller pools of the peptides such as those responsible for intranuclear vasopressin and oxytocin release. Further evidence that intranuclear release of vasopressin and oxytocin is a prerequisite for cAMP stimulation of vasopressin and oxytocin release was obtained by demonstrating that d(CH2)5-D-Tyr(Me)VAVP, a potent combined V1a/V2/oxytocin receptor antagonist blocked stimulation of vasopressin and oxytocin release by 8-Br-cAMP.

Substance P and NPY differentially potentiate ATP and adrenergic stimulated vasopressin and oxytocin release.

The supraoptic nuclei are innervated by the A1 neurons of the caudal ventrolateral medulla. Substances colocalized in the A1 terminals include norepinephrine (NE), substance P (SP), ATP, and neuropeptide Y (NPY). ATP, acting at P(2x) receptors, caused rapid and unsustained stimulation of vasopressin (VP) and oxytocin (OT) release from perifused explants of the hypothalamo-neurohypophysial system. SP elicited a concentration-dependent stimulation of VP and OT release that was large and sustained compared with other stimuli. ATP, but not phenylephrine (PE, alpha(1)-adrenergic agonist), augmented the response to SP (1 microM). In contrast, NPY did not alter basal nor ATP-induced VP or OT release, but it did cause sustained potentiation of PE-induced VP and OT release. The Y(1)-agonist, [Leu(31),Pro(34)]-NPY, increased VP and OT release, suggesting that the ineffectiveness of NPY reflects opposing actions at pre- and postsynaptic receptors. However, [Leu(31),Pro(34)]-NPY did not potentiate hormone responses to ATP or PE. The differential responses to these colocalized neurotransmitters and neuropeptides illustrate the range of potential responses that stimulation of this pathway might elicit from supraoptic neurons.

The role of steroid hormones in the regulation of vasopressin and oxytocin release and mRNA expression in hypothalamo-neurohypophysial explants from the rat.

Vasopressin and oxytocin release from the neural lobe, and the vasopressin and oxytocin mRNA contents of the supraoptic and paraventricular nuclei are increased by hypertonicity of the extracellular fluid. The factors regulating these parameters can be conveniently studied in perifused explants of the hypothalamo-neurohypophysial system that include the supraoptic nucleus (but not the paraventricular nucleus) with its axonal projections to the neural lobe. Vasopressin and oxytocin release and the mRNA content of these explants respond appropriately to increases in the osmolality of the perifusate. This requires synaptic input from the region of the organum vasculosum of the lamina terminalis. Glutamate is a likely candidate for transmitting osmotic information from the organum vasculosum of the lamina terminalis to the magnocellular neurones, because agonists for excitatory amino acid receptors stimulate vasopressin and oxytocin release, and because increased vasopressin release and mRNA content induced in hypothalamo-neurohypophysial explants by a ramp increase in osmolality are blocked by antagonists of both NMDA (N-methyl-D-aspartate) and non-NMDA glutamate receptors. Osmotically stimulated vasopressin release is also blocked by testosterone, dihydrotestosterone, oestradiol and corticosterone. Both oestrogen and dihydrotestosterone block NMDA stimulation of vasopressin release, and in preliminary studies oestradiol blocked AMPA stimulation of vasopressin release. Thus, steroid inhibition of osmotically stimulated vasopressin secretion may reflect inhibition of mechanisms mediated by excitatory amino acids. Recent studies have demonstrated numerous mechanisms by which steroid hormones may impact upon neuronal function. Therefore, additional work is warranted to understand these effects of the steroid hormones on vasopressin and oxytocin secretion and to elucidate the potential contribution of these mechanisms to regulation of hormone release in vivo.

Effect of age and testosterone on the vasopressin and aquaporin responses to dehydration in Fischer 344/Brown-Norway F1 rats.

To determine if the aging-associated decline in testosterone results in attenuated vasopressin (VP) responses to dehydration, testosterone implants were given to aged male Fischer 344Brown-Norway F1(F344BNF1) rats. Water deprivation caused comparable dehydration, increased plasma VP (pVP), and decreased posterior pituitary (PP) VP content in 4-, 15-, and 28-month-old rats. Dehydration increased VP mRNA content of supraoptic nuclei only at 4 months, whereas VP mRNA length was increased at both 4 and 15 months of age. The elevated pVP in the water-deprived aged rats indicates that even without an increase in VP mRNA content, PP VP storage was adequate to maintain elevated pVP. Dehydration increased aquaporin-2 content at 4, but not at 15 or 28 months of age, suggesting decreased renal responsiveness to VP. Testosterone replacement did not produce dehydration-induced increases in VP mRNA or aquaporin-2. Therefore, testosterone deficiency does not result in altered VP responses to dehydration in aged F344BNF1 rats.

Purinergic and adrenergic agonists synergize in stimulating vasopressin and oxytocin release.

The A1 catecholamine neurons of the caudal ventrolateral medulla transmit hemodynamic information to the vasopressin (VP) neurons in the hypothalamus. These neurons corelease ATP with norepinephrine. Perifused explants of the hypothalamoneurohypophyseal system were used to investigate the role of these substances on VP release. ATP (100 micrometer) increased VP release 1.5-fold (p = 0.027). The response was rapid but unsustained. It was blocked by the P(2) receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). The alpha(1)-adrenergic agonist phenylephrine (PE; 100 micrometer) also increased VP release by 1.5-fold (p = 0.014). Again, the response was rapid and unsustained. However, simultaneous perifusion of explants with ATP (100 micrometer) and PE (100 micrometer) resulted in a threefold to fourfold increase in VP release, which was sustained for as long as 4 hr. There was a similar synergistic effect of ATP and PE on oxytocin release. Interestingly, the synergistic response was delayed approximately 40 min relative to the response to either agent alone. Several experiments were performed to elucidate the cellular mechanisms of this synergism. The effect was blocked by PPADS, a protein kinase C inhibitor (bisindolylmaleimide I HCl), and actinomycin, an inhibitor of gene transcription. These data suggest that P(2X) receptor activation, PKC-mediated phosphorylation, and gene transcription are required for the synergistic response. The marked synergism of these coreleased agents is probably important to achieve sustained increases in plasma VP in response to prolonged hypotension. These observations may also have broad applications to CNS function, because ATP may be coreleased at noradrenergic synapses throughout the CNS.

Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization.

Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non-specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo-neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of non-n-methyl-d-aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7-dinitroquinoxaline-2,3-dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P <0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low-Mg2+ or Mg2+-replete medium. However, blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 microM) caused a marked increase in VP release in response to 100 microM glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 mM glutamate. These results support a role for non-NMDA receptor activation in osmotic regulation of VP release.

N-methyl-D-aspartic acid stimulation of vasopressin release: role in osmotic regulation and modulation by gonadal steroids.

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N-methyl-D-aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo-neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK-801 (10 microM) and AP5 (100 microM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1 microM NMDA in Mg2+-replete compared with 5 microM in low-Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50 pg/ml) and DHT (3 ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.

Expression of floor plate in dispersed mesencephalic cultures: role in differentiation of tyrosine hydroxylase neurons.

The availability of sufficient numbers of dopaminergic neurons for transplantation has been an important issue. Recently, it has been shown that the ventral floor plate (FP4-positive) cells and the transcription factor HNF-3beta are important in the signals that terminate proliferation and produce differentiation of the dopaminergic phenotype. In this study, dispersed mesencephalon from embryonic rats at Day 11 postcoitus (E-11), 1 day prior to the birth of TH cells, were cultured for 48 h and 1 week to evaluate TH neuronal differentiation and/or proliferation in vitro. The number of TH cells increased 14x between 48 h and 1 week in culture. In dispersed E-14 cultures, the presence of FP4 and HNF-3beta markers was demonstrated using immunohistochemistry. The majority of FP4-positive cell clusters were associated with TH neurons, suggesting that floor plate cells may have participated in TH neuron differentiation in culture. Antisense oligonucleotide probe for HNF-3beta mRNA added daily to cultured E-14 cells blocked the HNF-3beta expression, but had no effect on the FP4 or TH expression. These studies suggest a potentially important role for floor plate cells in the differentiation of TH cells, and differentiation and/or proliferation of TH cells in dispersed cultures of E-11 is demonstrated.

Effect of aging on vasopressin and aquaporin responses to dehydration in Fischer 344-brown-Norway F1 rats.

The plasma vasopressin (VP) response to 72 h of water deprivation is attenuated in 30-mo-old Fischer 344 (F344) rats relative to 4-mo-old rats. This appears to reflect an inability to increase VP synthesis. In contrast, elevated plasma VP has been reported in the Brown-Norway (BN) strain of rats secondary to reduced renal VP responsivity. The response to dehydration in the F1 cross of these strains (F344BNF1) was evaluated. Male rats, 4 and 30 mo old, were deprived of water for 72 h or allowed water ad libitum. In response to dehydration, plasma sodium and hematocrit were significantly increased in both young and aged rats (P < 0.05), but plasma VP, urine osmolality, and aquaporin 2 expression were only increased in the young rats (P < 0.05). Posterior pituitary content of immunoreactive VP was depleted in the young but not the old rats. Thus the aged F344BNF1 rats demonstrated a deficit in VP release in response to an apparently similar dehydration stimulus. This deficit was different from those previously reported for either the F344 or BN strains. Thus further studies are required to determine the abnormalities underlying this response.