Cloning of neuromedin B and its receptor in the rabbit and generating a polyclonal antibody to the neuromedin B protein.

Neuromedin B (NMB) is a highly conserved bombesin-related neuropeptide found in mammals. Neuromedin B (NMB) executes its effect by binding to the cell surface receptor, neuromedin B receptor (NMBR). In this study, we cloned the rabbit NMB and NMBR genes. The similarity and phylogenetic analyses of NMB and NMBR gene sequences were performed. The expression of NMB and NMBR mRNA in the rabbit was investigated using real-time RT-PCR. Our bioinformatic analysis demonstrated that the cloned rabbit NMB precursor cDNA encoded Gly-His-Phe-Met-NH2 amino acids at the C-terminus, and that its receptor possessed typical transmembrane features. The NMB mRNA was highly expressed in the CNS, while the NMBR mRNA was widely expressed in many tissues, with the highest expression in the gastrointestinal tract. The studies on the NMB distribution and function are limited by the lack of a specific antibody to this neuropeptide. In this paper, polyclonal NMB antibody was generated in mice. Western blotting analysis revealed that the prepared antibody could specifically recognize the recombinant and the endogenous NMB proteins. Immunohistochemistry analysis indicated that the NMB protein was localized in the cytoplasm of the pituitary cells. The existence of NMB protein in the hypothalamic-pituitary-gonadal axis suggests that NMB might function in rabbit reproduction.

Incongruent pattern of neurokinin B expression in rat and mouse brains.

The expression patterns of Tac2 and NK3 mRNA and of pep2, the neurokinin B (NKB) precursor protein, were compared in rats and mice. Pep2 immunoreactivity was observed in fibers, terminals, and perikarya in the brains of both species, but the number of NKB-immunoreactive cells was generally smaller in mice than in the corresponding nuclei in rats. Congruent distribution patterns of Tac2 mRNA and NKB were found in many nuclei of the thalamus and hypothalamus (habenula, anterodorsal nucleus, preoptic area, arcuate nucleus, paraventricular nucleus). However, mice expressed Tac2 mRNA neither in the hippocampus nor in the nucleus of the lateral olfactory tract, in contrast to rats. Accordingly, mice showed no NKB in the projection areas of these nuclei, such as the olfactory tubercle, whereas a clear NKB signal was present in rat tissues. Surprisingly, we found nearly identical NK3 mRNA expression patterns in both species, despite the species differences in NKB expression. Thus, although the expression patterns of Tac2 and NKB are similar in rats and mice, noteworthy differences exist. Our results have important implications for the interpretation of behavioral results concerning the NKB/NK3 system in these species.

A tachykinin-like factor increases glutamate toxicity in rat cerebellar granule cells.

Tachykinins (TKs), which include substance P, neurokinin A and neurokinin B, constitute a group of neuropeptides widely expressed in the CNS where they play several functions connected with neural modulation often in synergy with glutamate excitatory transmission. The aim of this study was to assess whether TKs modulate glutamate response of in vitro cultured cerebellar granule neurons and whether GSA (glutamate-sensitizing activity), a peptide released by these neurons, belongs to the TKs family. Treatment with substance P and other neurokinin 1 receptor (NK1) agonists does not affect the response of cerebellar granule neurons to glutamate toxicity. On the contrary, agonists neurokinin 2 receptor (NK2) and neurokinin 3 receptor (NK3) agonists increase, in a dose and time dependent fashion, the response of the same neurons to glutamate. MEN 10,627, a selective NK2 receptor antagonist, and (Trp(7),betaAla(8)) NKA (4-10), a selective NK3 receptor antagonist inhibit not only the sensitizing action to glutamate of their respective agonists. These antagonists almost equally reduce the glutamate-sensitizing activity of GSA. Such activity is also abolished in the presence of a polyclonal antibody directed against neurokinin B (NKB). These findings indicate that TKs increase glutamate sensitivity in cerebellar granule neurons and that the GSA previously detected in conditioned media of the same cultured neurons belongs to the TK family although its primary structure as compared to known TKs remains to be established.

Arginine-vasopressin neurons in the rat hypothalamus produce neurokinin B and co-express the tachykinin NK-3 receptor and angiotensin II type 1 receptor.

Secretion of arginine-vasopressin (AVP) from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei is induced by neurokinin B (NKB) and angiotensin. To characterize the mechanisms by which this occurs, we used immunohistochemical techniques to assess the ability of AVP-producing neurons to express NKB, NKB receptor (NK-3 receptor) and angiotensin II type 1 receptor (AT-1 receptor). Double fluorescence immunohistochemistry indicated that AVP-immunoreactive cell bodies in the PVN and SON, as well as their axon varicosities in the posterior pituitary, co-express NKB. Almost all AVP-neuron perikarya also expressed both the NK-3 receptor and AT-1 receptor. Thus, AVP-producing neurons in the PVN and SON, which are regulated by NKB, are themselves a source of NKB. Furthermore, the regulation of AVP release by these neurons by NKB and angiotensin II is mediated by the NK-3 receptor and the AT-1 receptor, respectively.

Differential modulation of human immunoglobulin isotype production by the neuropeptides substance P, NKA and NKB.

The modifying effects of tachykinins substance P, neurokinin A and neurokinin B on immunoglobulin production were analyzed in an in vitro culture system. Purified human T- and B-cells were stimulated with TGFbeta2 and IL-5 to induce preferential IgA production. Neuropeptides had the following effects. (1) The levels of IgA and IgG4 production were enhanced by IL-5 and TGFbeta2; IgA levels remained constant or were slightly augmented by neuropeptides, whereas IgG4 was further augmented. (2) IL-5 and TGFbeta2 did not alter IgG3 production, but neuropeptides stimulated secretion of this subclass. (3) IgG1 and IgM production were inhibited by IL-5 and TGFbeta2. This effect was prevented by neuropeptides. (4) Other isotypes including IgG2 and IgE remained unaffected. Except for IgM, these effects were blocked by specific receptor antagonists indicating specificity. The tachykinin receptor NK-1 mRNA was detected in B- and T-cells, whereas NK-3 mRNA was only present in T- and B-cell coculture following activation. Furthermore, neuropeptide effects depended on cytokine co-stimulation and the presence of T-cells. These results suggest that neuropeptides are potent modifiers of preferential IgA synthesis.

Neurokinin B- and substance P-like immunoreactivity are co-localized in enteric nerves of rat ileum.

The tachykinins (TKs) substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) have conserved C-terminal sequences and mediate similar physiological responses by activating neurokinin receptors found on neural and smooth muscle cells. Many enteric nerves express preprotachykinin A (PPT A) mRNA and synthesize SP and NKA. However, it is unclear if NKB is synthesized in enteric neurons as many antibodies developed against NKB also recognize other TKs. Therefore, the cellular distribution of NKB-like-immunoreactivity (NKB-ir) in rat ileum was examined using selective antisera raised against either synthetic Cys10-NKB or peptide 2 (P2), a non-tachykinergic peptide sequence in NKB precursor protein. NKB-ir and P2-ir had a similar distribution in varicose nerve fibers in submucosal and myenteric ganglia and almost all ganglia contained immunoreactive nerves. Few submucosal or myenteric neuronal somata contained strong immunoreactivity. Preabsorption of NKB or P2 antisera with their respective cognate peptides, but not with other TK peptides, abolished specific immunostaining. Finally, co-localization of NKB-/P2-ir with SP-ir suggested that most NKB-/P2-ir nerve fibers contain SP-ir, but some SP-ir nerves do not contain detectable NKB-/P2-ir. These results indicate that PPT B products P2 and NKB are localized in a subpopulation of enteric nerves containing TKs encoded by PPT A. Stimulation of these nerves may release NKB to activate local neurokinin receptors.

Morphologic maturation of tachykinin peptide-expressing cells in the postnatal rabbit retina.

Tachykinin (TK) peptides, which include substance P, neurokinin A, two neurokinin A-related peptides and neurokinin B, are widely present in the nervous system, including the retina, where they act as neurotransmitters/modulators as well as growth factors. In the present study, we investigated the maturation of TK-immunoreactive (IR) cells in the rabbit retina with the aim of further contributing to the knowledge of the development of transmitter-identified retinal cell populations. In the adult retina, the pattern of TK immunostaining is consistent with the presence of TK peptides in amacrine, displaced amacrine, interplexiform and ganglion cells. In the newborn retina, intensely immunostained TK-IR somata are located in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL). They are characterized by an oval-shaped cell body originating a single process without ramifications. TK-IR processes are occasionally observed in the IPL and in the outer plexiform layer (OPL). Long TK-IR fiber bundles are observed in the ganglion cell axon layer. TK-IR profiles resembling small somata are rarely observed in the INL adjacent to the OPL. At postnatal day (PND) 2, some TK-IR cells display more complex morphologic features, including processes with secondary ramifications. Long TK-IR processes in the IPL are often seen to terminate with growth cones. Between PND 6 and PND 11 (eye opening), there is a dramatic increase in the number of immunolabeled processes with growth cones both in the IPL and in the OPL and the mature lamination of TK-IR fibers in laminae 1, 3 and 5 of the IPL is established. TK-IR cells attain mature morphological characteristics and the rare, putative TK-IR somata in the distal INL are no longer observed. After eye opening, growth cones are not present and the pattern typical of the adult is reached. These observations indicate that the development of TK-IR cells can be divided into an early phase (from birth to PND 6) in which these cells establish their morphological characteristics, and a later phase (from PND 6 to eye opening) in which they are involved in active growth of their processes and likely in synapse formation. Since TK peptides are thought to play neurotrophic actions in the developing nervous system and they are consistently present in the retina throughout postnatal development, they may also act as growth factors during retinal maturation.

Role of neuromedin B in the in vitro thyrotropin release in response to thyrotropin-releasing hormone from anterior pituitaries of eu-, hypo-, and hyperthyroid rats.

A role of neuromedin B (NB), a bombesin-like peptide, as an inhibitory paracrine/autocrine regulator of thyrotropin secretion has been suggested. We previously reported (10) that basal thyroid-stimulating hormone (TSH) release in vitro was decreased by NB and increased in the presence of a highly potent antiserum against NB (aNB). In these experiments, we studied the effects of NB (10(-11) - 10(-7) M) and antiserum against NB (aNB, 1:2000 dilution) on basal TSH release and the response to thyrotropin-releasing hormone (TRH) (0.5 x 10(-8) M) from incubated anterior pituitaries from eu-, hypo-, and hyperthyroid rats. As expected, in euthyroid rats NB decreased basal and TRH-stimulated TSH release, but only at the highest concentration tested (10(-7) M). Incubation of the pituitaries from euthyroid rats with the antiserum against NB increased basal TSH release above that from glands of normal rabbit serum-incubated controls, as anticipated based on the concept that NB inhibits TSH release from the pituitary glands of euthyroid animals. The antiserum did not augment the response to TRH, suggesting that NB released in this situation, although suppressing basal release, had no effort on the stimulated release induced by TRH. Glands from hypothyroid rats had a slightly lower basal TSH release and decreased response to TRH than glands from euthyroid rats. They responded with a decrease in basal TSH release at a much lower concentration of NB (10(-9) M) than pituitaries from euthyroid animals. Surprisingly, pituitaries from hypothyroid rats showed a paradoxical increased release of TSH in response to the lowest concentration of NB (10(-11) M), which decreased with increasing concentrations and was not distinguishable from control release in the presence of TRH at the highest concentration of NB (10(-7) M). We hypothesize that the increased responsiveness to the inhibition of basal TSH release by NB in the hypothyroid pituitaries may be related to an upregulation of NB receptors in this situation, in which the release of NB is diminished because of loss of feedback via thyroid hormones. The view that NB secretion was reduced in the hypothyroid situation was supported by the fact that there was no change in TSH release or the response to TRH following treatment with aNB in these animals. Remarkably, in the glands from the hyperthyroid rats, although basal TSH secretion was significantly lower than that from euthyroid pituitaries and response to TRH was also decreased, NB (10(-11)-10(-7) M) instead of decreasing TSH release augmented it significantly. Also, the response to TRH was significantly augmented but only at the lowest concentration of NB tested (10(-11 M). That NB was probably being secreted in vitro from the hyperthyroid pituitaries was indicated by an increased basal TSH release as well as a higher TSH medium concentration after TRH in the presence of the aNB. These results support the concept that the glands from the hyperthyroid animals secrete more NB because of positive feedback of thyroid hormones directly on the thyrotropes to increase NB synthesis and release which downregulates NB receptors on the gland. This downregulation of receptors in some manner reverses the inhibitory action of NB on basal and TRH-stimulated TSH release. In conclusion, the results provide further evidence for an important role of NB as an autocrine regulator of TSH release, which is modulated by increased release of NB induced by thyroid hormones.

A study of tachykinin-immunoreactivity in the cat visual cortex.

The localization of tachykinin-immunoreactivity in the cat visual cortex (area 17) was investigated using immunohistochemical methods. Strong laminar specificity was observed, with immunoreactivity highest in layer V, followed by layers I, VI, II and III, and the lowest density in layer IV. Most of the immunoreactive product was localized in neuronal processes. A few immunopositive cell bodies were also present. The immunopositive neurons were non-pyramidal, multipolar, or bipolar in shape, and mostly found in layer V. There were particularly dense immunopositive fibers and varicosities around somata in layer V. These may represent tachykinin-containing presynaptic terminals (boutons). The results provide anatomical evidence that tachykinins may primarily affect layer V neurons in the cat visual cortex.

Distribution of neurokinin A-like and neurokinin B-like immunoreactivity in human peripheral tissues.

Using specific radioimmunoassay and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of the two peptides in human peripheral tissues were studied. Both NKA-like immunoreactivity (NKA-LI) and NKB-like immunoreactivity (NKB-LI) were present in the walls of the gut and gall bladder and in the pancreas. In the gut, the values for NKA-LI were 0.56-35.73 pmol/g wet weight, while those in pancreas and gall bladder were 0.64-0.68 and 0.36 pmol/g wet weight, respectively. The values of NKB-LI were 0.45-2.66 pmol/g wet weight in the gut, 0.93-1.65 pmol/g wet weight in the pancreas, and 0.30 pmol/g wet weight in the gall bladder. The immunocytochemical reactivity to both peptides was localized to ganglia of the submucosal and myenteric nerve plexuses in the gut wall, and to neurons in the muscle layer and mucosa of the gut wall. Weak but positive NKA-LI appeared in nerve cells of the pancreas, while NKB-LI was not detectable in the pancreas. Conversely, in the gall bladder wall, NKA-LI was undetectable while a very faint NKB-LI was found in the muscle layer. The localization of NKA corresponded closely to that of NKB in the tissues although the relative concentrations of the peptides varied from organ to organ.

Neurokinin-immunoreactivity in human neuroblastomas. Evidence for selective expression of the preprotachykinin (PPT) II gene.

Factors regulating differentiation of the peripheral nervous system (PNS) have been widely studied in neuroblastomas which are tumors of the PNS. Five neuroblastomas were investigated for their content of tachykinin neuropeptides, which arise from two distinct genes which appear differentially expressed in the PNS. Radioimmunoassay and column chromatography revealed large amounts of neurokinin B in three of these tumors and the absence of substance P, neurokinin A, neuropeptide K and neuropeptide gamma from all five tumors. This suggests that neuroblastomas can selectively express the preprotachykinin (PPT) II gene and that they may be valuable for investigating the factors involved in the regulation of these two structurally-related neuropeptide genes.

Synthesis and immunological evaluation of N-terminal, noncrossreactive tachykinin antigens.

The N-terminal hexa- or pentapeptide sequences of the three mammalian tachykinins substance P, neurokinin A, and neurokinin B have been synthesized by the conventional solid-phase procedure with 6-aminocaproyl-S-(acetamidomethyl)cysteine as a C-terminal spacer and attachment function. A fourth sequence, with an additional N-terminal 6-aminocaproyl residue on the substance P-hapten sequence, was cyclized N- to C-terminally. For this purpose, a four-level protection scheme has been applied: BOC-TFA for N-terminal protection and cleavage; TFA-stable but HF-labile anchoring function and side-chain protection; S-acetamidomethyl for semipermanent thiol protection. The side chain amino function of Lys was protected with NO2Z, stable against HF but readily cleaved with hydrogenation. The hapten sequences were coupled to maleimidated BSA, after the Acm group was removed by mercury/hydrogen sulfide treatment. Mice immunized with the three linear hapten sequences produced sera that were specific in enzyme-linked immunosorbant assay for the presented hapten and the respective tachykinin but displayed no crossreactivity at all toward the other haptens nor to one of the other tachykinins. It is concluded that this approach produced antisera, specific and selective for its respective mammalian tachykinins.