We investigated the effect of bypassing (using dexamethasone, DEX) or inhibiting (using carbenoxolone, CBX) feto-placental 11b-HSD in rats, on subsequent offspring HPA axis regulation and stress-induced behaviour. Pregnant Wistar rats were injected with CBX, DEX or vehicle daily throughout pregnancy. A separate group received DEX injections in the last week of pregnancy only (DEX3), as this had previously been shown to contain the critical time window for programming of hypertension and hyperglycaemia. All treatments reduced birth weight (CBX 12%, DEX1-3 15%, DEX3 7%). This was reversed by 4 and 6 weeks of life in DEX3 and CBX offspring, respectively, while offspring of dams treated with DEX throughout pregnancy had reduced body weight throughout life (7% at 5 months of age). Prenatal exposure to glucocorticoids resulted in increased basal corticosterone (CORT) levels (CBX 0.70 mg/dl, DEX3 0.76 mg/dl, control 0.40 mg/dl) and a trend (p=0.07) towards increased corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) in adult offspring. In addition, CBX offspring had reduced PVN glucocorticoid receptor (GR) mRNA and a prolonged stress response. Hippocampal GR and mineralocorticoid receptor (MR) mRNA expression was reduced only in DEX3 offspring, which also showed increased CRH mRNA levels in PVN. CBX and DEX3 rats displayed impaired non-spatial learning. In addition, exploratory behaviour in an open field was affected by prenatal glucocorticoid exposure, especially in DEX3 offspring, which showed reduced locomotion and rearing. The behavioural alternations are associated with increased expression of MR, GR and CRH mRNAs in the amygdala, a structure implicated in the expression of fear and anxiety. These data suggest that disturbance of the feto-placental enzymatic barrier to maternal glucocorticoids, or <i>in utero</i> exposure to DEX, reduces birth weight and produces permanent subtle alterations of the HPA axis combined with behavioural inhibition or impaired coping in aversive situations.

No description available.

Adrenal corticosteroids play a major role in the negative feedback control of the hypothalamic-pituitary-adrenal axis, which ensures that corticosteroid levels are optimal for homeostatic adaptation. The objective of this thesis was to investigate the validity of a model of early glucocorticoid (GC) feedback inhibition of adrenocorticotrophic hormone (ACTH) secretion developed through the study of mouse corticotroph tumour (AtT20) cells. Early GC inhibition was found to require the synthesis of new mRNA and proteins. Previous work indicated that calmodulin is one of the GC-induced proteins that may mediate the inhibitory action of GC on ACTH secretion. Therefore, AtT20 cells that constitutively over-expressed chicken calmodulin were established to test if elevated calmodulin level could mimic GC action. Corticotrophin releasing factor (CRF)-stimulated ACTH secretion by stably transferred AtT20 cells that showed elevated calmodulin mRNA and protein levels and wild type AtT20 cells was found not be significantly different. Events downstream to the <I>de novo</I> synthesis of proteins in GC feedback inhibition were studied in cultured rat anterior pituitary cells. Membrane depolarisation elicited by a combination of a depolarising concentration of KC1 with 8-(4-chlorophenylthio)adenosine-3',5'-cyclic-monophosphate (CPT-cAMP) or CRF antagonised the GC inhibition of stimulated ACTH secretion. Since the control of membrane potential involves K<SUP>+</SUP>-channels, the effect of K<SUP>+</SUP>-channel blockers on GC inhibition was tested. The BK-channel inhibitor, charybdotoxin, had no significant effect on GC inhibition, nor did other common K<SUP>+</SUP>-channel blockers (tetraethylammonium, apamin and 4-aminopyridine). Clofilium (IsK-type K<SUP>+</SUP>-channel blocker) and astemizole (an antihistamine with anti-HERG-type K<SUP>+</SUP>-channel properties) were found to significantly reduce the inhibition of the CRF response by GC. More specific blockers of IsK-type K<SUP>+</SUP>-channels (chromanol 293B, WAY-123398) or HERG-type K<SUP>+</SUP>-channels (E4031), dofetilide) had no significant effect on GC inhibition. Previous studies have also suggested that control of intracellular cAMP levels via Ca<SUP>2+</SUP>-dependent feedback is important in GC inhibition. Hence, it was tested if clamping of cAMP at high levels could alter GC inhibition. High cAMP levels were induced and sustained using arginine vasopressin (AVP) or rolipram (inhibitor of cAMP-dependent cyclic nucleotide phosphodiesterase) in combination with CRF.

Study of the haemodynamic response to drugs is important in evaluation of their ability to achieve reduction of oesophageal variceal bleeding and to optimise their haemodynamic action in patients with liver disease. My studies have concentrated on the evaluation of two drugs, N-acetylcysteine and isosorbide-5-mononitrate (a drug reported to have a problem of tolerance). The portal and systemic haemodynamic response to low and high dose isosorbide-5-mononitrate (Is-5-Mn) was studied. 10 mg or 40 mg of oral Is-5-Mn was given acutely and chronically (bd for 4 weeks) allowing a 16 hour nitrate free interval to 25 patients with cirrhosis. Both doses of nitrate reduced the hepatic venous pressure gradient acutely and chronically, without evidence of tolerance by a reduction in the wedged hepatic venous pressure. The effect on mean asygos blood flow was dependent on the initial azygos blood flow, not on the dose of nitrate used; if low then vasodilation was seen in response to Is-5-Mn and vice-versa. As the effect of Is-5-Mn on cardiac output, systemic resistance and its pharmacokinetics in the presence of chronic liver disease has been previously reported this was not studied further. Intravenous N-acetylcysteine (NAC) was given to 11 patients with cirrhosis. There was no effect on mean heart rate or blood pressure despite a significant fall in systemic and pulmonary vascular resistance. Cardiac index increased but estimated liver blood flow and hepatic venous pressure gradients did not change significantly. Administration of NAC increased oxygen delivery but not arteriovenous oxygen extraction ratio or mean tissue oxygen consumption. Therefore NAC administration does not appear to confer haemodynamic benefit on patients with cirrhosis.

Cyclic nucleotide hydrolysis by phosphodiesterases (PDEs) is an important process determining the amplitude and duration of the responses to cAMP generating agonists. In adenohypophysial corticotrophs and the mouse pituitary corticotroph tumour AtT20 cells, ACTH secretion is known to be mediated through the cAMP pathway. The aim of this thesis is to identify the cAMP-hydrolysing phosphodiesterase isozymes expressed in rat adenohypophysis and AtT20 cells, and to study the regulation of their activities by cAMP-dependent pathway in AtT20 cells. Multifaceted analysis showed that Ca<SUP>2+</SUP>/calmodulin (CaM)-stimulated (PDE1) and cAMP-specific, rolipram-inhibitable (PDE4) PDE isotypes are expressed in AtT20 cells as well as rat adenohypophysis. RT-PCR analysis revealed the expression of mRNAs from the PDE1B, PDE1C, PDE4A, PDE4B and PDE4D subfamilies in AtT20 cells and PDE1C, PDE4B and PDE4D in rat adenohypophysis. Stimulation of cAMP pathway in AtT20 cells was found to enhance the activity of PDE4 and to reduce that of PDE1. Calyculin A, an inhibitor of protein phosphatase 1/2A, synergistically enhanced the effect of CPT-cAMP on PDE1 and had an additive effect on PDE4. Incubation of AtT20 cytosol with the catalytic subunit of PKA produced the same changes of PDE1 and PDE4 activities as the activation of PKA in intact cells. The reduction of PDE1 activity was associated with a markedly diminished response to Ca<SUP>2+</SUP>/CaM stimulation. Immunoprecipitation studies with isozyme-specific antibodies showed that PDE4D isozymes accounted for more than half of the control as well as CPT-cAMP and/or calyculin A activated PDE4 activity. Immunoblot analysis of the immunoprecipitate showed two distinct bands that on the basis of apparent molecular weights correspond to the PDE4D3 and PDE4D5 splice variants. The migration of both bands on SDS-PAGE were retarded upon CTP-cAMP and/or calyculin A treatments, which is consistent with the reported effect of PKA phosphorylation on PDE4D3. In sum, these data indicate that 1) Both Ca<SUP>2+</SUP>/CaM-dependent (PDE1) and Ca<SUP>2+</SUP>/CaM-independent (primarily PDE4) PDEs are expressed in rat adenohypophysis and AtT20 cells; 2) Low K<SUB>m</SUB> PDE1, plausibly PDE1C, is found in rat adenohypophysis as well as AtT20 cells; 3) The rapid and differential regulation of PDE1 and PDE4 by cAMP-dependent phosphorylation and dephosphorylation in AtT20 cells provides a means for fine tuning the Ca<SUP>2+</SUP>/CaM dependency and the extent of cAMP hydrolysis in relation to the momentary status of cAMP and Ca<SUP>2+</SUP>/CaM pathways.

To investigate agonist-induced internalisation of the VPAC<SUB>2</SUB> receptor we generated a C-terminally epitope-tagged receptor (VPAC<SUB>2</SUB>-HA) and mutant receptors with serial C-terminal truncations. Addition of the epitope tag had no significant effect on the second messenger signalling or agonist binding properties of the VPAC<SUB>2</SUB> receptor stably expressed in the human embryonic kidney cell line (HEK293). Iodinated helodermin, a VPAC<SUB>2</SUB> receptor agonist, was rapidly internalised in cells expressing the VPAC<SUB>2</SUB> and VPAC<SUB>2</SUB>-HA receptors following incubation at room temperature or at 37°C. Internalisation kinetics of the VPAC<SUB>2</SUB> and VPAC<SUB>2</SUB>-HA receptor were indistinguishable. In serum starved cells, the epitope-tagged VPAC<SUB>2</SUB> receptor was predominantly located in the plasma membrane. Treatment with VIP caused a marked shift in receptor distribution from the plasma membrane to a single intracellular site. Receptor internationalisation was dependent upon the concentration of agonist, incubation time and temperature. Removal of agonist resulted in the reappearance of receptor immunoreactivity at the plasma membrane; this movement was unaffected by the presence of the protein synthesis inhibitor cycloheximide, suggesting that the majority of receptors are recycled. Other work in our laboratory has demonstrated that truncation of the C-terminal intracellular domain of the VPAC<SUB>2</SUB> receptor prevents agonist-induced receptor phosphorylation. Nonetheless, truncated receptors were still able to internalise, indicating that phosphorylation is not an absolute requirement for VPAC<SUB>2</SUB> receptor internalisation. The truncated VPAC<SUB>2</SUB> receptor did not desensitise so we postulate that phosphorylation is necessary for this phenomenon. VPAC<SUB>2</SUB> receptors colocalise with transferrin receptors, which are internalised via clathrin pits and act as a marker of endosomes. No colocalisation was observed between the VPAC<SUB>2</SUB> receptor and a marker for the trans-golgi network, indicating that the receptor does not recycle through this compartment. This work provides the first direct evidence for agonist-induced internalisation of the VPAC<SUB>2</SUB> receptor.

The aim of this investigation was to examine the mechanisms implicated in the processes of learning and memory in the rodent hippocampus using electrophysiological recording techniques to monitor CA1 pyramidal cell activity. In the presence of ionotropic glutamate and g-aminobutyric acid (GABA) receptor antagonists, stimulation of the septohippocampal cholingeric afferents, in <i>stratum oriens,</i> produced a slow excitatory postsynaptic potential (EPSP). Subsequent pharmacological classification established that this response was mediated by activation of muscarine acetylcholine receptors (mAChR) and as such this response was termed an EPSP_M. Specific blockers of voltage-gated Ca²⁺channels, w-conotoxin GVIA and w-agatoxin IVA, revealed that the release of ACh necessary to evoke the EPSP_M was mediated by activation of both N- and P/Q-type Ca²⁺channels. Blockade of presynaptic 4-AP-senstivie K⁺ channels further enhanced the release of ACh. A previous report had shown that mAChR-mediated synaptic transmission could be modulated by adenosine A₁ receptor activation but had not examined the precise cellular mechanisms underlying this effect. Investigations have revealed that the activation of A₁ receptors inhibited the EPSP_M irrespective of the Ca²⁺channel supporting this response and that there maybe a partial involvement of 4-AP sensitive K⁺ channels. As adenosine A₁ receptors are known to act via the G-protein, G_i/o, we also investigated the involvement of cAMP in the inhibition of the EPSP_M. It was demonstrated that forskolin stimulated increases in cAMP partially occluded and 8-Br cAMP application fully occluded the adenosine A₁ receptor-mediated inhibition of the EPSP_M. Most synapses are under the regulation of a variety of GPCRs. In this respect we also demonstrated that opioid receptor agonists could modulate the EPSP_M. Interestingly, it was found that opioid agonists acting at a presynaptic m-opioid receptor caused an enhancement of the EPSP_M although the mechanism of how this is achieved is unclear.

Receptors for the neuropeptides VIP and PACAP belong to a novel sub-family of G protein-coupled receptors, the secretin/calcitonin/parathyroid hormone receptor family. The rat VIP<SUB>2</SUB> receptor was recently cloned in this laboratory and the present project was carried out to characterise the signalling mechanisms used by this receptor and define the role of crucial receptor domains. These studies have primarily involved the transient expression of receptors in host cells. The results demonstrated for the first time that both the VIP<SUB>1</SUB> and VIP<SUB>2</SUB> receptors can stimulate phospholipase C (PLC) in addition to adenylyl cyclase (AC) and that this PLC stimulation occurs by a pertussis toxin(PTx)-sensitive mechanism. Corresponding GTPγS modulation of ligand-binding to the VIP<SUB>2</SUB> receptor in COS7 cell membranes was shown to be partially PTx-sensitive, suggesting an interaction of the receptor with a PTx-sensitive G protein. An epitope-tagged human VIP<SUB>2</SUB> receptor was expressed in COS7 cells and immunoprecipitated with its associated G proteins for Western-blotting studies. Immunoreactivity for Gα<SUB>q</SUB>, Gα<SUB>s</SUB> and a member of the Gα<SUB>i/o/t/z </SUB>family, other than Gα<SUB>i1 </SUB>or Gα<SUB>i2</SUB>, appeared to be associated with the receptor. The use of specific calcium channel blockers identified a role for receptor-mediated calcium influx in VIP<SUB>2</SUB> receptor-mediated PLC stimulation. The PACAP and VIP<SUB>2</SUB> receptors contain many common structural features but have their own distinct pharmacological profiles. The function of specific domains of the VIP<SUB>2</SUB> receptor was investigated by creating PACAP/VIP<SUB>2</SUB> receptor chimaeric constructs and C-terminal truncations of the VIP<SUB>2</SUB> receptor. These constructs allowed identification of the primary region of the receptor responsible for ligand-binding and the probable site of interaction with a PTx-sensitive G protein and also assessment of any contribution of the C-terminus to AC and PLC stimulation.

Intracellular recordings were made from presumptive serotonergic neurones in the <i>in vitro</i> slice preparation of the rat dorsal raphe nucleus. The modulation of excitability of presumptive serotonergic neurones by 5-HT was investigated. Confirming previous observations bath-application of 5-HT led to a membrane hyperpolarisation with an associated decrease in input resistance. The reversal level was approximately - 90 mV, a value close to that predicted for a potassium-mediated event. The 5-HT-evoked hyperpolarisation was blocked by WAY-100635, a result consistent with the response being mediated by the 5-HT_1A receptor subtype. WAY-100635 alone was found to have no effect on membrane potential or input resistance in this study and in this sense is silent. The blockade of the 5-HT_1A-evoked hyperpolarisation with WAY-100635 revealed a 5-HT-evoked depolarisation associated with an increase in cell excitability. In the majority of cells tested there was no significant change in input resistance and I/V analysis failed to show a point of intersection in the range -120mV to -60mV. However, some cells did show a change in input resistance and a point of intersection could be observed at approximately -50mV. The depolarising response was observed in the presence of TTX showing it to be independent of propagated action potentials and thus unlikely to involve network synaptic events. In the present of WAY-100635 the 5-HT₂ agonist DOI evoked a depolarisation with similar properties to that evoked by 5-HT. The more selective 5-HT_2B/2C agonist mCPP and the 5-HT_1B agonist CP 93129 had no effect in WAY-100635 treated cells. The broad spectrum 5-HT receptor antagonist methysergide blocked the 5-HT-evoked depolarisation in 6 of 8 cells as well as the depolarising action of DOI. The 5-HT₂ antagonist ketanserin completely blocked the 5-HT-evoked depolarisation, as did the 5-HT_2Aselective antagonist MDL 100927.

Current therapy for PD is focused on dopamine replacement by the use of L-DOPA and dopamine agonists, which relieve the Parkinsonian motor symptoms led by the degeneration of dopaminergic neurons in the substantia nigra par compacta (SNc). However, the long-term use of these agents could induce side effects and fail to slow down the progression of the nigral dopaminergic neuronal loss. Thus, there is an increasing demand for neuroprotective agents that can relieve the symptoms and alter the dopaminergic neuronal death in PD. Excitotoxicity, due to the excessive glutamate release from subthalamic nucleus (STN) to the SNc, could jeopardize the neurodegeneration in the SNc. Thus, the modulation of the glutamate could prevent the cellular loss in PD SNc. Accordingly, the selective ligands acting on metabotropic glutamate receptors (mGluRs) were proved to be anti-Parkinsonian in PD models. Furthermore, the group III mGluR agonist, L-(+)-2-amino-4-phosphonobutyric acid (L-AP4), was proved to be neuroprotective in the 6-OHDA PD model. In this study, therefore, the subtypes of group III mGluRs, such as mGluR4, 6, 7 and 8, was examined in order to provide a more specific neuroprotection. Thus, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) and (S)-3,4-dicarboxyphenylglycine ((S)-DCPG) were utilized for the activation of mGluR4 and 8 respectively which are expressed prevalently in basal ganglia. Herein, the concentration-dependent and receptor mediated neuroprotection of PHCCC and (S)-DCPG against 6-OHDA toxicity in rodent model of PD were demonstrated. Furthermore, PHCCC and (S)-DCPG are neuroprotective when administered to animal systemically. Moreover, the neuroprotective potential of PHCCC and (S)-DCPG were putatively demonstrated in another PD model of lactacystin, an ubiquitin-proteasome inhibitor. In this study, we also reported the effect of PHCCC and (S)-DCPG on the glial cells involved in the PD models, elucidating the underlying neuroprotective mechanism of these ligands. Thus, mGluR4 and 8 may be a promising therapeutic target for further pharmacological development.