Distribution, targeting, and internalization of the sst4 somatostatin receptor in rat brain.

Somatostatin mediates its diverse physiological effects through a family of five G-protein-coupled receptors (sst(1)-sst(5)); however, knowledge about the distribution of individual somatostatin receptor proteins in mammalian brain is incomplete. In the present study, we have examined the regional and subcellular distribution of the somatostatin receptor sst(4) in the rat CNS by raising anti-peptide antisera to the C-terminal tail of sst(4). The specificity of affinity-purified antibodies was demonstrated using immunofluorescent staining of HEK 293 cells stably transfected with an epitope-tagged sst(4) receptor. In Western blotting, the antiserum reacted specifically with a broad band in rat brain, which migrated at approximately 70 kDa before and approximately 50 kDa after enzymatic deglycosylation. sst(4)-Like immunoreactivity was most prominent in many forebrain regions, including the cerebral cortex, hippocampus, striatum, amygdala, and hypothalamus. Analysis at the electron microscopic level revealed that sst(4)-expressing neurons target this receptor preferentially to their somatodendritic domain. Like the sst(2A) receptor, sst(4)-immunoreactive dendrites were often closely apposed by somatostatin-14-containing fibers and terminals. However, unlike the sst(2A) receptor, sst(4) was not internalized in response to intracerebroventricular administration of somatostatin-14. After percussion trauma of the cortex, neuronal sst(4) receptors progressively declined at the sites of damage. This decline coincided with an induction of sst(4) expression in cells with a glial-like morphology. Together, this study provides the first description of the distribution of immunoreactive sst(4) receptor proteins in rat brain. We show that sst(4) is strictly somatodendritic and most likely functions in a postsynaptic manner. In addition, the sst(4) receptor may have a previously unappreciated function during the neuronal degeneration-regeneration process.

Allelic and somatic variations in the endogenous opioid system of humans.

People with a genetic predisposition for substance abuse have defects in genes for the opioid peptides and receptors. A high number of polymorphisms have been detected in the mu-opioid receptor, some of which result in pharmacological alterations. The opioid peptide proopiomelanocortin proved extraordinarily rich in mutations that often lead to severe phenotypical consequences. Prodynorphin displays a polymorphic regulation of transcription. Variants of the mu- and the delta-opioid receptor showed positive associations with opiate and/or alcohol addiction in some studies. However, these associations were weak, indicating a small contribution of the opioid system to these disorders.

Effects of opioid antagonists and morphine in a hippocampal hypoxia/hypoglycemia model.

The influence of opioid antagonists and of morphine on rat hippocampal slices in a model of reversible hypoxia/hypoglycemia was investigated by assessment of evoked field potentials (population spike amplitude). In control slices, a brief hypoxia/hypoglycemia led to a loss of field potentials followed by an impaired recovery (40-50% of baseline) during reperfusion. In contrast, restoration was significantly improved when the opioid receptor antagonists funaltrexamine (mu) or naltrindole (delta) were administered prior to and during hypoxia/hypoglycemia. In addition, recovery was improved in brain slices derived from mu-opioid receptor-deficient mice as compared to wild-type mice, indicating a deleterious role of endogenous opioids in hypoxia/hypoglycemia. Exogenous opiate exposure with morphine (0.1, 1.0, 10 microM) prior to hypoxia/hypoglycemia caused a slight concentration dependent increase of evoked field potentials. When morphine exposure was terminated after 1h and immediately followed by hypoxia/hypoglycemia, an impaired recovery of population spike amplitude was obtained, dependent on morphine concentration during preincubation. These results demonstrate that morphine aggravates neurotoxic effects of hypoxia/hypoglycemia. Conversely, when onset of hypoxia/hypoglycemia was delayed for 3h after morphine termination, a significantly improved recovery was observed. Similarly, in vivo administration of morphine 12h prior to slice preparation resulted in a dose dependent improved recovery of field potentials after hypoxia/hypoglycemia. These results provide evidence that preconditioning with morphine is able to induce neuroprotective effects.

Effect of Brn-3a deficiency on primary nociceptors in the trigeminal ganglion.

Immunohistochemistry for substance P, somatostatin and vanilloid receptor subtype 1 as well as receptors for somatostatin and opioids was performed on the trigeminal ganglion in wild-type and Brn-3a knockout mice at postnatal day 0. In wild-type mice, the trigeminal ganglion contained abundant substance P-, vanilloid receptor subtype 1-, sst2A receptor- and delta-opioid receptor-immunoreactive neurons, while the ganglion had only a few mu-opioid receptor-immunoreactive neurons. The Brn-3a deficiency had an effect on the cell size but not the number of substance P-immunoreactive neurons. In knockout mice, the proportion of small immunoreactive neurons markedly increased and that of medium- to large-sized immunoreactive ones correspondingly decreased (mean +/- S.D. = 54.7 +/- 29.1 microm2, range = 10.9-220.8 microm2) compared to wild-type mice (mean +/- S.D. = 116.6 +/- 58.6 microm2, range = 27.3-400.7 microm2). As for vanilloid receptor subtype 1-immunoreactive neurons, the number and cell size was barely affected by the deficiency. On the other hand, the loss of Brn-3a caused a decrease in the number of sst2A receptor- or delta-opioid receptor-immunoreactive neurons (more than 95% reduction) and an increase in the number of mu-opioid receptor-immunoreactive neurons (9.3-fold increase). Somatostatin-immunoreactive neurons were not detected in the trigeminal ganglion of wild-type or mutant mice at postnatal day 0. The present study suggests that Brn-3a deficiency may have effects on the survival of trigeminal nociceptors and their expression of some neurochemical substances.

Delta-opioid receptor-immunoreactive neurons in the rat cranial sensory ganglia.

Immunohistochemistry for delta-opioid receptor (DOR) was performed on the rat cranial sensory ganglia. The immunoreactivity was detected in 16%, 19% and 11% of neurons in the trigeminal, jugular and petrosal ganglia, respectively. The nodose ganglion was devoid of such neurons. DOR-immunoreactive (IR) neurons were mostly small to medium-sized (trigeminal, range = 62-851 microm(2), mean +/- SD = 359 +/- 175 microm(2); jugular, range = 120-854 microm(2), mean +/- SD = 409 +/- 196 microm(2); petrosal, range = 167-1146 microm(2), mean +/- SD = 423 +/- 233 microm(2)). Double immunofluorescence method revealed that all DOR-IR neurons were also immunoreactive for calcitonin gene-related peptide. The cutaneous and mucosal epithelia in the oro-facial region, tooth pulp, taste bud and carotid body were innervated by DOR-IR nerve fibers. In the brainstem, IR nerve terminals were located in the superficial medullary dorsal horn and dorsomedial part of the subnucleus oralis as well as the solitary tract nucleus. The present study suggests that DOR-IR neurons may be associated with nociceptive and/or chemoreceptive function in the cranial sensory ganglia.

Immunohistochemical determination of five somatostatin receptors in meningioma reveals frequent overexpression of somatostatin receptor subtype sst2A.

Meningioma is one of a variety of human tumors that exhibit a very high density of somatostatin receptors and in many cases show a true positive somatostatin receptor scintigraphy. However, the level of expression of individual somatostatin receptor proteins in meningioma has not been investigated. We have recently developed a panel of somatostatin receptor subtype-specific antibodies that effectively stain formalin-fixed, paraffin-embedded tumor tissue (S. Schulz et al., Clin. Cancer Res., 4: 2047-2052, 1998). In the present study, we have used these antibodies to determine the somatostatin receptor status of 40 randomly selected meningiomas. Immunoreactive staining for all somatostatin receptors was clearly located at the plasma membrane of the tumor cells and completely blocked with antigenic peptide. The vast majority of tumors (29 cases; 70%) were positive for sst2A immunoreactivity; among these, 20 (69%) tumors showed high levels of sst2A immunoreactivity. In contrast, all other somatostatin receptors were only detected sporadically, and none of these cases revealed a particularly strong staining. However, it is uncertain to what extent somatostatin receptor-immunoreactive staining intensity may translate into somatostatin receptor protein expression on the tumor cells. Therefore, in a prospective study, 16 surgically removed meningiomas were collected, and the level of sst2A expression was determined using Western blot analysis. Whereas sst2A was readily detectable as a broad band migrating at Mr 70,000 in 12 (75%) of these tumors, 8 tumors (50%) showed particularly high levels of immunoreactive sst2A receptors. There was an excellent correlation (P < 0.001) between the level of sst2A protein expression detected in Western blots and the sst2A- immunoreactive staining seen in tissue sections. Thus, the frequent overexpression of the sst2A receptor may explain the high tracer uptake often observed in meningioma patients during somatostatin receptor scintigraphy. Moreover, this simple immunohistochemical method could prove useful in identifying those cases of recurrent disease that may possibly respond to therapy with sst2-selective agonists.

Immunocytochemical detection of somatostatin receptors sst1, sst2A, sst2B, and sst3 in paraffin-embedded breast cancer tissue using subtype-specific antibodies.

The long-acting somatostatin analogue octreotide (SMS 201-995) inhibits growth of certain breast cancer cell lines in vivo and in vitro. Because the antiproliferative action of octreotide depends on at least the presence of somatostatin receptors, it is crucial to determine the pattern of somatostatin receptor protein expression on the tumor cells. In the present study, we have raised polyclonal antibodies to somatostatin receptor subtypes (ssts) sst1, sst2A, sst2B, and sst3 using peptides corresponding to their COOH-terminal sequences. These antisera were used for immunocytochemical staining of paraffin sections of 33 primary breast cancers. Somatostatin receptor-like immunoreactivity (Li) was predominantly localized to the plasma membrane of the tumor cells. In the vast majority of positively stained tumors, somatostatin receptor-Li was uniformly present on nearly all tumor cells. Both the level and the pattern of expression of ssts varied greatly between individual carcinomas. sst2A-Li and/or sst2B-Li was detectable in 28 tumors (85%); among these, 14 tumors (42%) showed particularly high levels of sst2-Li. sst1-Li was found in 17 (52%) cases and sst3-Li in 16 (48%) cases. The expression of ssts was independent of patient age, menopausal status, diagnosis, histological grade, and levels of estrogen and progesterone receptors. The immunocytochemical determination of somatostatin receptor status allows direct detection of receptor protein on the tumor cells and, hence, may provide more precise information than reverse transcription-PCR for predicting response to octreotide therapy in breast cancer.

Proenkephalin B (prodynorphin)-derived opioid peptides: evidence for a differential processing in lobes of the pituitary.

The distribution of peptides derived from the novel opioid peptide precursor proenkephalin B (prodynorphin) was studied in lobes of the pituitary with antibodies against alpha-neoendorphin (alpha-neo-E) beta-neoE, dynorphin (DYN)-(1-17), DYN-(1-8), and DYN B in combination with gel filtration and high pressure liquid chromatography. In the posterior pituitary, all five opioid peptides occurred in high and about equimolar concentrations, whereas putative precursor peptides were found in only minor quantities. In contrast, in the anterior pituitary immunoreactive (ir-) DYN-(1-17) and ir-DYN B consisted exclusively of a common precursor species with a mol wt of about 6000. Six thousand-dalton DYN may be comprised of the C-terminal portion of proenkephalin B, with the sequence of DYN-(1-17) at its N-terminus. Moreover, the major portions of ir-alpha-neo-E and ir-beta-neoE in the anterior pituitary were found to be of an apparent mol wt of 8000. These findings indicate a differential processing of the opioid peptide precursor proenkephalin B in the two lobes of the pituitary. The anterior pituitary seems to process proenkephalin B predominantly into high mol wt forms of neo-E and DYNs, whereas in the posterior pituitary proenkephalin B undergoes further proteolytic processing to the smaller opioid peptides alpha-neo-E, beta-neo-E, DYN-(1-17), DYN-(1-8), and DYN B. Thus, processing differences may enable the selective liberation of different (opioid) peptides with distinct biological properties from one precursor within different tissues.

Postnatal development of beta-endorphin-related peptides in rat anterior and intermediate pituitary lobes: evidence for contrasting development of proopiomelanocortin processing.

The concentration of immunoreactive (ir) beta-endorphin (beta-END) in the neurointermediate pituitary lobe was 15-fold higher in adult than in newborn rats; in contrast, that of ir-beta-END in the anterior lobe was twice as high in newborn as in adult animals. Ir-beta-END in the neurointermediate lobe of newborn rats consisted exclusively of beta-END-sized peptides, indicating that at birth rats are capable of processing the opioid peptide precursor proopiomelanocortin (POMC) to beta-END. Moreover, beta-END-related peptides in the neurointermediate lobe of newborn rats were found to be predominantly alpha-N-acetylated and, therefore, inactivated with respect to their opiate-like properties. Further analysis of these alpha-N-acetylated forms on high performance liquid chromatography indicated that newborn rats predominantly contained alpha-N-acetyl-(Ac-)beta-END-(1-31), whereas the major forms in adult rats were Ac-beta-END-(1-27) and -(1-26). Thus, the C-terminal processing of Ac-beta-END-(1-31) to -(1-27) and -(1-26) may not yet be fully active at birth, in contrast to the processing of POMC to beta-END. In the anterior lobe of newborn rats, however, the ratio of beta-lipotropin/beta-END resembled that of adults, and more than 80% of beta-END-sized ir-material was found to consist of nonacetylated (and therefore opiate-active) beta-END-(1-31), as in adults, suggesting that the enzymatic system responsible for processing of POMC to beta-lipotropin and beta-END is already mature at birth. The high concentrations of beta-END in the anterior lobe of newborn rats suggest a possible role of this opioid peptide in perinatal development and/or parturition.

Chronic haloperidol treatment increases the level of in vitro translatable messenger ribonucleic acid coding for the beta-endorphin/adrenocorticotropin precursor proopiomelanocortin in the pars intermedia of the rat pituitary.

Chronic treatment of rats with haloperidol (1.5 mg/kg, once daily) over a period of 7--21 days resulted in a 80--100% increase in the tissue levels of immunoreactive beta-endorphin and in the in vitro release of immunoreactive beta-endorphin from the neurointermediate pituitary. Incorporation of [3H]phenylalanine into isolated neurointermediate pituitaries of haloperidol-treated rats revealed an increase in the amount of label incorporated into the beta-endorphin/ACTH precursor proopiomelanocortin (POMC) to a similar extent (about 80%) but had essentially no effect on the conversion of the precursor into beta-lipotropin and beta-endorphin. Extraction of messenger (m) RNA from neurointermediate pituitaries followed by cell-free translation in a reticulocyte system showed an increase in the total level of translatable mRNA (about 25%). The content of translatable mRNA coding for POMC, however, was increased by 100-150%. Time-course studies revealed a parallelism between the effect of haloperidol on the level of in vitro translatable mRNA coding for POMC and the ability of the drug to increase the concentrations of beta-endorphin in the neurointermediate pituitary. A complete reversal of the effects of haloperidol was seen 2 weeks after discontinuation of the drug. These findings suggest that the chronic blockade of dopaminergic receptors by haloperidol causes a reversible increase in the beta-endorphin biosynthesis in the rat intermediate pituitary at the pretranslational level by markedly increasing the level of translatable mRNA coding for POMC.

Calcium ion and cyclic adenosine 3',5'-monophosphate regulate proopiomelanocortin messenger ribonucleic acid levels in rat intermediate and anterior pituitary lobes.

The role of the second messengers cAMP and Ca++ in the control of proopiomelanocortin (POMC) gene expression was investigated with the use of hybridization with cloned complementary DNA probes. The effects of cAMP-related drugs on POMC messenger RNA (mRNA) levels were assessed in primary cultures of intermediate (IL) and anterior rat pituitary cells maintained in serum-free medium. 8-Bromo-cAMP (1 mM), but not 8-bromo-cGMP (1 mM), induced a 2-fold increase in IL and anterior lobe cell after 2 days of treatment. A similar increase was obtained with the adenylate cyclase-activating drugs forskolin (1 microM) and cholera toxin (100 ng/ml) or the phosphodiesterase inhibitor RO 20-1724 (100 microM). At 48 h, all these treatments had increased beta-endorphin accumulation in the medium and transiently decreased the cellular beta-endorphin content in IL cells, suggesting a parallel effect of cAMP-related drugs on secretion and biosynthesis. Incubating the cells with the Ca++ channel antagonists D600 (50 microM), verapamil (50 microM), and the dihydropyridine nifedipine (0.1 microM) decreased basal POMC mRNA levels, whereas the dihydropyridine BAYK 8644 (0.1 microM), which activates the Ca++ channel, increased POMC mRNA levels after 2 days. In addition, nifedipine decreased the stimulatory effect of forskolin, whereas BAYK 8644 further stimulated the forskolin-increased POMC mRNA levels in IL cells. We conclude that both Ca++ and cAMP may regulate the gene expression of POMC.

Ketoconazole inhibits corticotropic cell function in vitro.

The effects of ketoconazole (KC) on secretion and biosynthesis of ACTH and generation of cAMP in rat anterior pituitary cells were investigated in vitro. KC inhibits CRF-stimulated ACTH release from rat anterior pituitary fragments in a dose-dependent fashion between 1.5 and 100 microM. The effect of CRF as a releaser of ACTH was fully restored after KC was removed from the medium. Similar effects were observed in primary cultures of rat anterior pituitary cells. KC dose-dependently decreased basal and CRF-stimulated ACTH release. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with KC. The effects of KC on ACTH release and biosynthesis seem to be mediated by cAMP, since KC inhibits basal and CRF-stimulated cAMP release and content within the same dose range. Since the stimulatory effects of cholera toxin, sodium fluoride, and forskolin were dose-dependently inhibited by KC and since the addition of (Bu)2cAMP abolished the inhibiting effect of KC, it is concluded that KC acts by inhibition of the catalytic component of the adenylate cyclase holoenzyme.

Nitroimidazole derivatives inhibit anterior pituitary cell function apparently by a direct effect on the catalytic subunit of the adenylate cyclase holoenzyme.

Nitroimidazole derivatives dose-dependently decreased basal and CRF-stimulated ACTH release, basal and GRF-stimulated rat GH release, and basal rat PRL release in primary cultures of rat anterior pituitary cells. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with the nitroimidazole derivatives. Miconazole, econazole, isoconazole, clotrimazole, and bifonazole had similar or more pronounced effects on anterior pituitary function compared to ketoconazole, whereas metronidazole and etomidate were less effective. The positive correlation between the number of phenylated side-chains or phenolic rings of the imidazole molecule and the efficacy to inhibit activity on pituitary hormone secretion suggests a structure-activity relationship of these compounds. The effects of the nitroimidazole derivatives on anterior pituitary hormone release and biosynthesis were mediated by cAMP. Thus, basal and CRF-, cholera toxin-, and forskolin-stimulated adenylate cyclase activities in rat anterior pituitary cell membranes determined by cAMP formation were suppressed by the nitroimidazole derivatives. Pertussis toxin did not diminish the nitroimidazole derivative effect on cAMP formation. The adenylate cyclase inhibitory effect of these substances was independent of the presence of GTP in the assay system, underlining a direct effect on the catalytic subunit. In addition, basal and forskolin-stimulated cAMP generation in membranes of S49 lymphoma cyc-variants, which lack a functional Gs protein, was efficiently suppressed (by up to 90%) by the nitroimidazole derivatives. In conclusion, ketoconazole and other nitroimidazole derivatives inhibit anterior pituitary hormone synthesis and secretion apparently by a direct effect on the catalytic subunit of the adenylate cyclase system.

The two isoforms of the mouse somatostatin receptor (mSSTR2A and mSSTR2B) differ in coupling efficiency to adenylate cyclase and in agonist-induced receptor desensitization.

The somatostatin receptor 2 (mSSTR2) is alternatively spliced into two isoforms (mSSTR2A and mSSTR2B) which differ at the C-terminus. Both receptors bind somatostatin peptides with a similar high affinity when stably expressed in CHO-K1 cells. However, the spliced form (mSSTR2B) mediates a more efficient inhibition of adenylate cyclase and is much more resistant to agonist-induced reduction of binding than the longer form (mSSTR2A). These findings indicate that alternative splicing may be a physiological mechanism to modulate receptor desensitization and G-protein coupling of mSSTR2.

Cloning and expression of an isoform of the rat mu opioid receptor (rMOR1B) which differs in agonist induced desensitization from rMOR1.

A novel rat mu opioid receptor (rMOR1B) has been isolated. It shows identity to the recently published sequence of rMOR1 [Chen, et al., Mol. Pharmacol., 44 (1993) 8-12] up to amino acid 386 and differs only in length and amino acid composition at the very carboxy-terminal tail. Both mu opioid receptor isoforms, when stably expressed in CHO-K1 cells, show similar affinities to opioid compounds and are equally effective in the inhibition of forskolin-induced cAMP formation. Reverse transcription polymerase chain reaction (RT-PCR) revealed that rMOR1B displays a similar distribution as rMOR1 in various rat brain areas. Studies measuring the inhibition of adenylate cyclase in cells that had been pre-exposed to the mu opioid agonist DAMGO indicated that rMOR1B is much more resistant to agonist-induced desensitization than rMOR1.

Regulation of mouse somatostatin receptor type 2 gene expression by glucocorticoids.

Somatostatin is a regulatory peptide with important inhibitory functions. Its effects are mediated by five receptors, sst1-sst5. Previous studies revealed that sst2 contains three alternative, tissue specific promoters. Here, molecular mechanisms for the regulation of sst2 transcription by glucocorticoids were investigated. Reporter gene assays in NG 108-15 cells and electrophoretic mobility shift assays revealed that a glucocorticoid responsive element at position -1044 on the second promoter mediates dexamethasone induction. These findings, the existence of a cAMP response element and the tissue dependent activity (brain, pituitary and gastrointestinal tissues) indicate the importance of the second sst2 gene promoter.

Identification of a functional 3',5'-cyclic adenosine monophosphate response element within the second promoter of the mouse somatostatin receptor type 2 gene.

Important physiological actions of somatostatin are mediated by the somatostatin receptor type 2. Its transcription is regulated by three tissue specific, alternative promoters. It is known that the mRNA of the somatostatin receptor type 2 gene is induced by cAMP, but little is known about the mechanisms underlying this regulation. We have identified and characterized a cAMP responsive element located at nucleotide -162 on the second promoter of the gene consisting of the classical palindromic octameric sequence 5'-TGACGTCA-3'. Using transient expression of reporter gene deletion constructs in NGC108-15 cells the necessity of the intact element for forskolin-induced reporter gene activity was demonstrated. The first and the third promoter are not responsive to forskolin, nor did any promoter respond to the phorbol ester PMA. Electrophoretic mobility shift assays in combination with competition experiments suggest the interaction of the promoter element with the cAMP responsive element binding protein.

Molecular cloning of a bovine MSH receptor which is highly expressed in the testis.

A novel G protein-coupled receptor (BDF3) was isolated from bovine genomic DNA by a combined approach of polymerase chain reaction (PCR) and hybridization techniques. The predicted amino acid sequence is 317 amino acids in length and displays 80% homology to the human alpha-MSH receptor MC1. Stably transfected into CHO-K1 cells, BDF3 mediates an increase of intracellular cAMP-levels following incubation with NLE-alpha-MSH, a potent alpha-MSH analog. The stimulation with ACTH1-10 is only moderate and gamma-MSH is ineffective. Northern blot analysis of bovine tissues revealed that the BDF3 gene is highly expressed in the testis as a single 2.3 kb mRNA species, suggesting an involvement of the BDF3 receptor in spermatogenesis.

Alternative promoter usage and tissue specific expression of the mouse somatostatin receptor 2 gene.

We have cloned the 5' upstream regulatory region of the mouse somatostatin receptor 2 gene. Its genomic organization is novel among all somatostatin receptor genes. It contains two previously unrecognized exons, separated by introns larger than 25 kb, and three tissue and cell specific alternative promoters. The first promoter in front of exon 1 is active only in AtT-20 tumor cells. The second promoter, located 5' to exon 2, is used in brain, pituitary, adrenals, pancreas, NG 108-15 and AtT-20 cells. Furthermore, it contains putative DNA elements for regulation by glucocorticoids, estradiol and cAMP. A third promoter, located in exon 3, is additionally used in lung, kidney and spleen.

Cloning of a novel putative G-protein-coupled receptor (NLR) which is expressed in neuronal and lymphatic tissue.

A novel G-protein-coupled receptor was isolated from mouse and rat neuronal and lymphatic tissues. The amino acid sequence of the rat receptor (rNLR) shows an overall homology of 80% to a recently cloned receptor from Burkitt's lymphoma cells (BLR1) which is exclusively expressed in lymphatic tissues [(1992) Eur. J. Immunol. 22, 2795]. Much less homology between rNLR and BLR1 was observed at the N-terminus (about 40%), whereas rNLR and the mouse homologue mNLR show 92% amino acid identity. Northern blot analysis of NLR revealed a predominant 5.5 kb mRNA species in various brain regions and neuronal cell lines, whereas in the spleen a 3 kb transcript is predominant. This distribution suggests a role of NLR in the nervous and immune systems.