Polymorphic variation as a driver of differential neuropeptide gene expression

The regulation of neuropeptide gene expression and their receptors in a tissue specific and stimulus inducible manner will determine in part behaviour and physiology. This can be a dynamic process resulting from short term changes in response to the environment or long term modulation imposed by epigenetically determined mechanisms established during life experiences. The latter underpins what is termed ‘nature and nurture, or ‘gene × environment interactions’. Dynamic gene expression of neuropeptides or their receptors is a key component of signalling in the CNS and their inappropriate regulation is therefore a predicted target underpinning psychiatric disorders and neuropathological processes. Finding the regulatory domains within our genome which have the potential to direct gene expression is a difficult challenge as 98% of our genome is non-coding and, with the exception of proximal promoter regions, such elements can be quite distant from the gene that they regulate. This review will deal with how we can find such domains by addressing both the most conserved non-exonic regions in the genome using comparative genomics and the most recent or constantly evolving DNA such as repetitive DNA or retrotransposons. We shall also explore how polymorphic changes in such domains can be associated with CNS disorders by altering the appropriate gene expression patterns which maintain normal physiology.     

Self-Sustaining Status Epilepticus: A Condition Maintained by Potentiation of Glutamate Receptors and by Plastic Changes in Substance P and Other Peptide Neuromodulators

Summary: We describe a model of self-sustaining status epilepticus (SSSE) induced by stimulation of the perforant path in free-running rats. In this model, seizures can be transiently suppressed by intrahippocampal injection of a blocker of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate synapses but return in the absence of further stimulation when the drug ceases to act. However, seizures are irreversibly abolished by blockers of N -methyl- d -aspartate receptors given locally or systemically. SSSE is enhanced by substance P and its agonists and blocked by its antagonists. SSSE induces novel expression of substance P-like immunoreactivity in hippocampal principal cells. These changes and those in other limbic peptides may contribute to the maintenance of SSSE and to the modulation of hippocampal excitability during epileptic seizures.     

Effects of haloperidol and clozapine on preprotachykinin-A messenger RNA, tachykinin tissue levels, release and neurokinin-1 receptors in the striato-nigral system.

The effects of haloperidol and clozapine on tachykinin tissue levels, preprotachykinin-A messenger RNA, spontaneous and potassium-evoked tachykinin release, dopamine D2 receptors, and [125I]Bolton-Hunter-substance P binding sites in the striato-nigral system were examined. Chronic administration (10 days) of the dopamine receptor antagonist haloperidol (2 mg/kg i.p.) significantly decreased tissue levels of substance P like-immunoreactivity and neurokinin A like-immunoreactivity in the striatum and the substantia nigra. The corresponding preprotachykinin-A mRNA was decreased in the striatum. Haloperidol did not affect the potassium-evoked tachykinin release in the substantia nigra but significantly increased the spontaneous release. Haloperidol increased the number of D2-receptors but left [125I]Bolton-Hunter-substance P binding sites, representing neurokinin 1 (NK-1) receptors, as determined by competition experiments with selective ligands, unchanged. Clozapine (30 mg/kg, i.m.) did not influence nigral and striatal tachykinin tissue levels, preprotachykinin-A mRNA and potassium-evoked release or spontaneous efflux in the substantia nigra, or D2-receptors and [125I]Bolton-Hunter-substance P binding sites. The present data indicate that neuroleptics influence the striato-nigral tachykinin system in different ways. Tachykinins may, therefore, contribute to the therapeutic and/or untoward effects of certain neuroleptic drugs.     

Organization and physiology of thesubstantia nigra

Summary A comprehensive review of the literature on the anatomy, electrophysiology and pharmacology of thesubstantia nigra is presented. A diagram is developed taking into account the interneuronal interactions of neurotransmitters and receptors that control firing rates and neurotransmitter releases. The central features of the diagram are a positive dopaminergic feedforward process and a positive feedback mechanism mediated by extrasynaptic substance P diffusing from striatal terminals to dopaminergic dendrites of thezona compacta neurons. This loop can enhance the transmission of information from thestriatum through thepars reticulata output neurons. The loop is controlled at the level of zona compacta neurons by a negative feedback supported by the dendritic release of dopamine and boosted by pedunculopontine activation mediated by muscarinic receptors. The output of the loop is controlled by two negative feedforward processes, both involving GABAergic striatonigral afferents. Application of the model to pharmacological studies of diverse behaviors including seizures, turning, and conditioned behaviors reveals unforseen relationships and may offer insights into, and directions for, further analysis of the mechanisms and functions involved.     

Human isolated small intestine: motor responses of the longitudinal muscle to field stimulation and exogenous neuropeptides

Summary (1) Longitudinal muscle strips from the human small intestine (jejunum/ileum) responded to electrical field stimulation (1–50 Hz) with frequency-related primary contractions which were largely atropine- (3 M) sensitive. When the tone was raised by addition of galanin (0.3 – 1 M), prostaglandin (PG) E₂ (1–10 M) or neurokinin A (NKA, 0.1 M), a frequency-related relaxation was evident which was potentiated by atropine. All the responses to field stimulation were abolished by tetrodotoxin (1 M), thus indicating their neural origin. (2) The atropine-sensitive primary contraction to field stimulation was virtually abolished by omega conotoxin fraction GVIA (CTX, 0.1–0.3 M) while the relaxations were CTX-resistant. The field stimulation-induced relaxations, which were observed in the presence of atropine and guanethidine (3 M), were also unaffected by apamin (0.1 M). (3) NKA and substance P (SP) produced a concentration- (1 nM–1 M for both peptides) related contraction, NKA being about 53 times more potent than SP. [Pro⁹]SP sulphone and [MePhe⁷]-NKB, selective agonists of the NK-1 and NK-3 receptor, respectively, were barely effective. On the other hand, [\Ala⁸]NKA(4–10), a selective NK-2 receptor agonist, had a potent contractile activity, similar to that of NKA. (4) Galanin (1 nM–1M) produced an atropine- and tetrodotoxin-resistant concentration-related contraction of longitudinal muscle of human isolated small intestine. The response to galanin did not show any sign of fading and was particularly suitable to study the evoked relaxations. (5) Calcitonin gene-related peptide (CGRP) (10–100 nM) consistently inhibited the nerve-mediated contractions of strips from the ileum while the effect on the jejunum was less pronounced. Vasoactive intestinal polypeptide (VIP, 0.1–1 M) inhibited nerve-mediated contractions both in the ileum and the jejunum. (6) These experiments indicate that both cholinergic excitatory and non-adrenergic non-cholinergic nerves affect motility of the longitudinal muscle of the human small intestine. Furthermore, several neuropeptides produce potent motor effects, the contractile response to tachykinins being apparently mediated by activation of NK-2 receptors.     

Differential effects of phosphoramidon and captopril on NK1 receptor-mediated plasma extravasation in the rat trachea

We sought to confirm the identity of the tachykinin receptor subtype that mediates plasma extravasation in the rat trachea, and assess the respective contributions of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) in regulating this tachykinin-induced response. To achieve these aims, we determined the relative potencies of several natural tachykinins and receptor-selective synthetic agonists, both before and after inhibiting NEP with phosphoramidon and ACE with captopril. We also determined the effects of these peptidase inhibitors, and the NK-1 receptor antagonist L-703,606, on the plasma extravasation produced by capsaicin, which releases tachykinins endogenously from sensory nerve endings. We found that the rank order of potency for producing plasma extravasation in the rat trachea was NK-1 receptor agonist ([Sar⁹, Met(O₂)¹¹] SP)>substance P>neurokinin A> neurokinin B. The NK-2 ([Nle¹⁰]NKA (4–10)) and NK-3 ([MePhe⁷]NKB) receptor agonists were without effect. We observed no change in the relative potencies of these peptides after giving rats phosphoramidon or captopril, which suggests that the different peptide potencies are not simply the consequence of different rates of enzymatic degradation. Nevertheless, the responses to substance P and neurokinin A were clearly potentiated in rats given phosphoramidon, indicating that NEP effectively degrades tachykininsin vivo. No significant potentiation was evident for any peptide in rats given captopril. Similarly, the plasma extravasation produced by capsaicin was potentiated in rats given phosphoramidon, but not in those given captopril. Pretreating rats with L-703,606 abolished the response to capsaicin. We conclude from these observations that NK-1 receptors mediate tachykinin-induced plasma extravasation in the rat trachea, and that NEP regulates this response with little or no contribution from ACE.     

Central Effects of Neuropeptide K on Water and Food Intake in the Rat

The present study investigated the effect on water and food intake in the rat of the intracerebroventricular (ICV) injection of neuropeptide K (NPK), the N-terminally extended form of neurokinin A. NPK inhibited water deprivation-induced water intake even at 31.2 ng/rat. At higher doses, it inhibited also water intake induced by ICV angiotensin II or by subcutaneous hypertonic NaCl, and food-associated drinking, the threshold dose being 125 ng/rat. In response to 125 ng/rat, food intake following 16 h food deprivation was not reduced. NPK inhibited food intake only at 500 ng/rat, a dose that evoked excessive grooming in treated animals. Thus NPK is a potent inhibitor of water deprivation-induced drinking and at higher doses it exerts a general antidipsogenic effect towards several dipsogenic determinants, without affecting food intake. On the other hand, it inhibits food intake only at high doses, 500 ng/rat or more, but this inhibition might be just related to the intense grooming evoked. The effects of NPK on ingestive behavior are markedly different from those of neurokinin A, which selectively inhibits osmotic drinking and food-associated drinking. These differences suggest that NPK itself may exert its effects on the central nervous system, not necessarily through the conversion to neurokinin A.     

Further evidence for multiple tachykinin receptors

Rank order potency data for substance P (SP), physalaemin and eledoisin have been determined in a variety of assays in vitro. On the guinea-pig field stimulated vas deferens, physalaemin was approximately eight and five times more potent than eledoisin and SP respectively whereas in the rat field-stimulated vas deferens, eledoisin was approximately five times more potent than either physalaemin or SP. It would appear that the guinea-pig preparation has a preponderance of 'P'-receptor subtypes, whereas the rat preparation contains mainly 'E'-receptor subtypes. We have attempted to validate the hypothesis that there are multiple tachykinin receptors. Experiments have been performed with the guinea-pig urinary bladder in vitro, using phenoxybenzamine (20 microM, 30 min) to alkylate receptor sites and using excess eledoisin (0.2-0.4 microM, 30 min) to protect receptors selectively against the effects of this agent. Our results showed that after pretreatment with phenoxybenzamine, the responses to eledoisin were significantly attenuated. Moreover, in the presence of excess eledoisin it was found that phenoxybenzamine was unable to produce any significant reduction of the subsequent responses. This difference between the responses to eledoisin and to other agonists suggests the existence of at least two different receptor subtypes for the tachykinins.     

NK-1 receptors mediate the tachykinin stimulation of salivary secretion: selective agonists provide further evidence

The relative contribution of NK-1, NK-2 and NK-3 receptors to the sialogogic response to i.v. tachykinins was investigated in urethane-anesthetized rats. [Pro⁹,Met(O₂)¹¹]substance P (SP), a selective NK-1 receptor agonist, was about 10 times more potent than SP itself and its action was unaffected by pretreatment. On the other hand [Nle¹⁰]neurokinin A (NKA)-(4–10) and [MePhe⁷]neurokinin B (NKB), two selective agonist for NK-2 and NK-3 receptors, respectively, were ineffective.     

NK1- and NK3-receptor mediated inhibition of 5-hydroxytryptamine release from the vascularly perfused small intestine of the guinea-pig

The effects of tachykinins on the spontaneous release of 5-hydroxytryptamine (5-HT) from the enterochromaffin cells into the portal circulation was investigated in vitro using the vascularly perfused isolated guinea-pig small intestine. 5-HT was determined by HPLC with electrochemical detection. Test substances were applied intraarterially. Substance P (SP) caused a concentration-dependent decrease in 5-HT outflow with an EC50 of 50pmol/l. Similarly, the selective NK1 receptor agonist SP methyl ester (1nmol/l) significantly inhibited 5-HT outflow (to 51±3%). When tetrodotoxin (1μmol/l) was added to the arterial perfusion medium, the inhibition by SP of 5-HT outflow was not affected. The selective NK1 receptor antagonist CP 99994 [(+)-(2S,3S)-3-(2-methoxyben-zylamino)-2-phenylpiperidine] (0.1μmol/l) prevented the inhibitory effect of SP (0.1μmol/l). Neither GR 94800 (PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-NleNH₂) (0.1μmol/l) nor SR 142801 [(S)-(N)-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenyl-piperidin-4-yl)-N-methylacetamide] (10nmol/l), which are selective NK2 and NK3 receptor antagonists, changed the SP-mediated inhibition. The selektive NK3 receptor agonist senktide (10nmol/l) also decreased the 5-HT outflow (to 57±5%). This inhibition was prevented by SR 142801 (10nmol/l) and by tetrodotoxin. CP 99994 (0.1μmol/l) significantly antagonized the senktide-mediated inhibition of 5-HT outflow. The outflow of 5-HT was unaffected when CP 99994, GR 94800 or SR 142801 alone were added to the perfusion medium. It is concluded that the release of 5-HT from enterochromaffin cells is directly inhibited by NK1 receptors, and indirectly by neuronal NK3 receptors whose stimulation leads to the release of SP. Received: 11 June 1997 / Accepted: 28 July 1997