Future pharmacological therapy in hypertension.

PURPOSE OF REVIEW: Hypertension (HTN) is a widespread and growing disease, with medication intolerance and side-effect present among many. To address these obstacles novel pharmacotherapy is an active area of drug development. This review seeks to explore future drug therapy for HTN in the preclinical and clinical arenas. RECENT FINDINGS: The future of pharmacological therapy in HTN consists of revisiting old pathways to find new targets and exploring wholly new approaches to provide additional avenues of treatment. In this review, we discuss the current status of the most recent drug therapy in HTN. New developments in well trod areas include novel mineralocorticoid antagonists, aldosterone synthase inhibitors, aminopeptidase-A inhibitors, natriuretic peptide receptor agonists, or the counter-regulatory angiotensin converting enzyme 2/angiotensin (Ang) (1-7)/Mas receptor axis. Neprilysin inhibitors popularized for heart failure may also still hold HTN potential. Finally, we examine unique systems in development never before used in HTN such as Na/H exchange inhibitors, vasoactive intestinal peptide agonists, and dopamine beta hydroxylase inhibitors. SUMMARY: A concise review of future directions of HTN pharmacotherapy.

Novel Medical Treatments for Hypertension and Related Comorbidities.

PURPOSE OF REVIEW: The purpose of this review is to summarize the most recent data available on advances in development of novel medical treatments for hypertension and related comorbidities. RECENT FINDINGS: Approximately half of all hypertensive patients have not achieved goal blood pressure with current available antihypertensive medications. Recent landmark studies and new hypertension guidelines have called for stricter blood pressure control, creating a need for better strategies for lowering blood pressure. This has led to a shift in focus, in recent years, to the development of combination pills as a means of achieving improved blood pressure control by increasing adherence to prescribed medications along with further research and development of promising novel drugs based on discovery of new molecular targets such as the counter-regulatory renin-angiotensin system. Fixed-dose combination pills and novel treatments based on recently discovered pathogenic mechanisms of hypertension that have demonstrated promising results as treatments for hypertension and related comorbidities will be discussed in this review.

Selective VIP Receptor Agonists Facilitate Immune Transformation for Dopaminergic Neuroprotection in MPTP-Intoxicated Mice.

Vasoactive intestinal peptide (VIP) mediates a broad range of biological responses by activating two related receptors, VIP receptor 1 and 2 (VIPR1 and VIPR2). Although the use of native VIP facilitates neuroprotection, clinical application of the hormone is limited due to VIP's rapid metabolism and inability to distinguish between VIPR1 and VIPR2 receptors. In addition, activation of both receptors by therapeutics may increase adverse secondary toxicities. Therefore, we developed metabolically stable and receptor-selective agonists for VIPR1 and VIPR2 to improve pharmacokinetic and pharmacodynamic therapeutic end points. Selective agonists were investigated for their abilities to protect mice against MPTP-induced neurodegeneration used to model Parkinson's disease (PD). Survival of tyrosine hydroxylase neurons in the substantia nigra was determined by stereological tests after MPTP intoxication in mice pretreated with either VIPR1 or VIPR2 agonist or after adoptive transfer of splenic cell populations from agonist-treated mice administered to MPTP-intoxicated animals. Treatment with VIPR2 agonist or splenocytes from agonist-treated mice resulted in increased neuronal sparing. Immunohistochemical tests showed that agonist-treated mice displayed reductions in microglial responses, with the most pronounced effects in VIPR2 agonist-treated, MPTP-intoxicated mice. In parallel studies, we observed reductions in proinflammatory cytokine release that included IL-17A, IL-6, and IFN-γ and increases in GM-CSF transcripts in CD4(+) T cells recovered from VIPR2 agonist-treated animals. Moreover, a phenotypic shift of effector to regulatory T cells was observed. These results support the use of VIPR2-selective agonists as neuroprotective agents for PD treatment. SIGNIFICANCE STATEMENT: Vasoactive intestinal peptide receptor 2 can elicit immune transformation in a model of Parkinson's disease (PD). Such immunomodulatory capabilities can lead to neuroprotection by attenuating microglial activation and by slowing degradation of neuronal cell bodies and termini in MPTP-intoxicated mice. The protective mechanism arises from altering a Th1/Th2 immune cytokine response into an anti-inflammatory and neuronal sparing profile. These results are directly applicable for the development of novel PD therapies.

Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery.

OBJECTIVE: We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries. METHODS: Concentration response curves to PACAP38, PACAP27, VIP and the VPAC(1) receptor agonist ([Lys15,Arg16,Leu27]-VIP[1-7]-GRF[8-27]) were constructed in the absence or presence of the PAC(1) receptor antagonist PACAP6-38 or the VPAC(1) receptor antagonist, PG97269. mRNA expression was measured using qPCR. RESULTS: PACAP38 was less potent than VIP in both arteries. Both peptides had lower potency and efficacy in meningeal than in coronary arteries, while mRNA expression of VPAC(1) receptor was more pronounced in meningeal arteries. PACAP6-38 reduced the E(max) of PACAP27, while PG97269 right-shifted the VIP-induced relaxation curve only in the coronary arteries. CONCLUSION: The direct vasodilatory effect of VIP and PACAP might be less relevant than the central effect of this compound in migraine pathogenesis.

Structure-activity relationship of vasoactive intestinal peptide (VIP): potent agonists and potential clinical applications.

Vasoactive intestinal peptide (VIP) has been identified as one of major peptide transmitters in the central and peripheral nervous systems, being involved in a wide range of biological functions. The general physiologic effects of VIP include vasodilation, anti-inflammatory actions, cell proliferation, hormonal secretion, regulation of gastric motility, and smooth muscle relaxation; therefore, VIP has emerged as a promising drug candidate for the treatment of several diseases. A number of clinical applications of VIP or its derivatives have been developed; however, VIP-based drugs are not yet in clinical use, possibly because of mainly two serious problems: (1) poor metabolic stability and (2) poor penetration to the desired site of action. To overcome these shortcomings, the development of efficacious VIP analogues and several drug delivery systems has been attempted on the basis of numerous structure-activity relationships (SAR) studies and pharmacological experiments. Combination of the use of potent VIP analogues and an appropriate drug delivery system might be advantageous for the VIP-based therapy. We review in this paper SAR studies of VIP for the identification of potent therapeutic agents, describe the development of selective and/or metabolically stable VIP receptor agonists/antagonists, and discuss the potential application for clinical treatment using drug delivery systems.

Bioactive analogues and drug delivery systems of vasoactive intestinal peptide (VIP) for the treatment of asthma/COPD.

Vasoactive intestinal peptide (VIP) is one of the major peptide transmitters in the central and peripheral nervous systems, being involved in a wide range of biological functions. In an airway system where VIP-immunoreactive nerve fibers are present, VIP acts as neurotransmitter or neuromodulator of the inhibitory non-adrenergic and non-cholinergic airway nervous system and influences many aspects of pulmonary biology. A clinical application of VIP has been believed to offer potential benefits in the treatment of chronic inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), however, its clinical application has been limited in the past for a number of reasons, including its extremely short plasma half-life after intravenous administration and difficulty in administration routes. The development of long-acting VIP analogues, in combination with appropriate drug delivery systems, may provide clinically useful agents for the treatment of asthma/COPD. In this review, development of efficacious VIP derivatives, drug delivery systems designed for VIPs and the potential application for asthma/COPD are discussed. We also include original data from our chemical modification experiments and formulation studies, which led to successful development of [R(15, 20, 21), L(17)]-VIP-GRR (IK312532), a potent VIP analogue, and a VIPs-based dry powder inhaler system.

Neuropeptide mimetics and antagonists in the treatment of inflammatory disease: focus on VIP and PACAP.

Corticosteroids are the mainstay treatment for most severe inflammatory disorders. Due to the considerable toxicity associated with their long-term use, there is a great need for alternative treatments. Recently, two closely related neuropeptides with potent neuromodulatory activities, vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) have emerged as candidate molecules for the treatment of such pathologies. These peptides act primarily on three high affinity receptor subtypes expressed on multiple immune cell types, and orchestrate a cytokine response that is primarily anti-inflammatory. In this regard, systemic treatment with these peptides has been shown to greatly reduce the clinical symptoms and alter the pathogenic and cytokine profiles in animal models of rheumatoid arthritis, Crohn's disease, septic shock, and multiple sclerosis. Likewise, VIP and PACAP receptor knockout and overexpressing mice show altered immune responses in different models. We review here data demonstrating the potential effectiveness of these peptides in immune disorders, discuss receptor pharmacology and signaling pathways, describe the development of receptor specific agonists and antagonists, and discuss pharmaceutical considerations relevant to the specific delivery of analogs to the appropriate targets.

The VIP-receptor system in neuroblastoma cells.

Neuroblastoma (NB), the most common extracranial tumor during childhood arises from the embryonic sympathetic nervous system. Remarkably, NB can spontaneously regress, even after metastasis, leading to complete remission. Subpopulations of neuroblastic (N-type) and nonneuronal cells coexist in NB. Expression of the high-affinity nerve growth factor (NGF) TrkA receptor in NB is correlated with good prognosis, while MYCN amplification is associated with advanced stages of disease. N-type cells undergo differentiation when treated with different compounds, such as retinoids, phorbol esters, growth and neurotrophic NGF and neuropeptides, especially vasoactive intestinal peptide (VIP). These substances stabilize proliferation, leading to a more mature neuronal phenotype, neurite outgrowth and induction of expression of sympathetic neuronal markers. Therefore, receptors for these substances and their associated signalling pathways, appear like promising targets for the development of novel NB therapeutics. The aim of the present review is to summarize the quite considerable array of data, concerning production of VIP and related peptides, expression of their receptors in NB and the key regulation exerted by the VIP-receptor system in the control of NB cell behaviour.

Vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide inhibit tumor necrosis factor-alpha production in injured spinal cord and in activated microglia via a cAMP-dependent pathway.

Tumor necrosis factor-alpha (TNF-alpha) production accompanies CNS insults of all kinds. Because the neuropeptide vasoactive intestinal peptide (VIP) and the structurally related peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) have potent anti-inflammatory effects in the periphery, we investigated whether these effects extend to the CNS. TNF-alpha mRNA was induced within 2 hr after rat spinal cord transection, and its upregulation was suppressed by a synthetic VIP receptor agonist. Cultured rat microglia were used to examine the mechanisms underlying this inhibition because microglia are the likely source of TNF-alpha in injured CNS. In culture, increases in TNF-alpha mRNA resulting from lipopolysaccharide (LPS) stimulation were reduced significantly by 10(-7) m VIP and completely eliminated by PACAP at the same concentration. TNF-alpha protein levels were reduced 90% by VIP or PACAP at 10(-7) m. An antagonist of VPAC(1) receptors blocked the action of VIP and PACAP, and a PAC(1) antagonist blocked the action of PACAP. A direct demonstration of VIP binding on microglia and the existence of mRNAs for VPAC(1) and PAC(1) (but not VPAC(2)) receptors argue for a receptor-mediated effect. The action of VIP is cAMP-mediated because (1) activation of cAMP by forskolin mimics the action; (2) PKA inhibition by H89 reverses the neuropeptide-induced inhibition; and (3) the lipophilic neuropeptide mimic, stearyl-norleucine(17) VIP (SNV), which does not use a cAMP-mediated pathway, fails to duplicate the inhibition. We conclude that VIP and PACAP inhibit the production of TNF-alpha from activated microglia by a cAMP-dependent pathway.

VPAC2-R mediates the lipolytic effects of pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide in primary rat adipocytes.

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are structurally and functionally related. Their actions have been shown to be mediated by three different receptor subtypes: PAC1-R, which has exclusive affinity for PACAP, and VPAC1-R and VPAC2-R, which have equal affinity for PACAP and VIP. We recently showed that PACAP38 induces lipolysis in rat adipocytes, and in the present study we examined whether VIP has similar effects and which of the three receptors mediates this PACAP/VIP action. We showed by RT-PCR that all three receptor subtypes are present in rat adipocytes. We demonstrated that VIP (1-100 nm), like PACAP38, stimulates lipolysis in isolated adipocytes, as determined by glycerol release. By a pharmacological approach, using antagonists and agonists specific for the receptor subtypes, we elucidated the mechanisms by which PACAP38 and VIP mediate their lipolytic effects. We found that antagonists of PAC1-R [PACAP(6-38)] and VPAC1-R (PG97-269) did not affect lipolysis induced by 0.1-100 nm PACAP38 or VIP, and that a VPAC1-R agonist [K15, R16, L27VIP(1-7)GRF(8-27)] did not affect lipolysis at 1-1000 nm. However, two different VPAC2-R agonists [Hexa-VIP(1-28) and Ro25-1553] clearly mimicked the lipolytic effect of PACAP38 and VIP. In addition, the VPAC2-R antagonist PG99-465 (100 nm) caused right-shifted dose-response curves of PACAP38- and VIP-induced lipolysis. These results therefore provide evidence that all three PACAP/VIP receptor subtypes are expressed in primary rat adipocytes, but that the VPAC2-R subtype is responsible for mediating the lipolytic effects induced by PACAP38 and VIP.

A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal: a potential therapy for type 2 diabetes.

Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) activate two shared receptors, VPAC1 and VPAC2. Activation of VPAC1 has been implicated in elevating glucose output, whereas activation of VPAC2 may be involved in insulin secretion. A hypothesis that a VPAC2-selective agonist would enhance glucose disposal by stimulating insulin secretion without causing increased hepatic glucose production was tested using a novel selective agonist of VPAC2. This agonist, BAY 55-9837, was generated through site-directed mutagenesis based on sequence alignments of PACAP, VIP, and related analogs. The peptide bound to VPAC2 with a dissociation constant (K(d)) of 0.65 nmol/l and displayed >100-fold selectivity over VPAC1. BAY 55-9837 stimulated glucose-dependent insulin secretion in isolated rat and human pancreatic islets, increased insulin synthesis in purified rat islets, and caused a dose-dependent increase in plasma insulin levels in fasted rats, with a half-maximal stimulatory concentration of 3 pmol/kg. Continuous intravenous or subcutaneous infusion of the peptide reduced the glucose area under the curve following an intraperitoneal glucose tolerance test. The peptide had effects on intestinal water retention and mean arterial blood pressure in rats, but only at much higher doses. BAY 55-9837 may be a useful therapy for the treatment of type 2 diabetes.

Effect of inactivating mutations on phosphorylation and internalization of the human VPAC2 receptor.

The VPAC(2) receptor, as all members of the G-protein-coupled receptor (GPCR)-B family, has two highly conserved motifs in the third intracellular (IC(3)) loop: a lysine and a leucine located at the amino-terminus and two basic residues separated by a leucine and an alanine at the carboxyl-terminus. This study evaluates the involvement of those conserved amino acid sequences in VPAC(2) signal transduction and regulation. The residues were mutated into alanine and mutants were expressed in Chinese hamster ovary (CHO) cells stably transfected with Galpha16 and aequorin. Mutation of L310 reduced efficacy of vasoactive intestinal polypeptide (VIP) to stimulate adenylate cyclase activity through Galphas coupling by 75%, without affecting VIP capability to stimulate an increase in [Ca(2+)](i) through Galpha16 coupling. Mutation of R325 and, to a lesser extend, K328 reduced VIP efficacy to stimulate [Ca(2+)](i) increase and VIP potency to stimulate adenylate cyclase. The combination of mutations of both amino- and carboxyl-terminus located conserved motifs of the IC(3) loop generates an inactive receptor with respect to [Ca(2+)](i) increase and adenylate cyclase activation, but also with respect to receptor phosphorylation and internalization that were indeed directly correlated with the potency of inactivation of the receptors. The amino-terminus of the VPAC(2) receptor IC(3) loop is thus involved in adenylate cyclase activation and the carboxyl-terminus of the IC(3) loop participates in both Galphas and Galpha16 coupling. The mutations studied also reduced both receptor phosphorylation and internalization in a manner that appeared directly linked to the alteration of Galphas and Galpha16 coupling.

Activation of the vasoactive intestinal peptide 2 receptor modulates normal and atrophying skeletal muscle mass and force.

Of the two known vasoactive intestinal peptide receptors (VPAC1R and VPAC2R), the VPAC2R is expressed in skeletal muscle. To evaluate the function of the VPAC2R in the physiological control of skeletal muscle mass, we utilized the VPAC1R selective agonist [K15,R16,L27]VIP(1-7) GRF(8-27)-NH2 and the VPAC2R selective agonist Ro-25-1553 to treat mice and rats undergoing either nerve damage-, corticosteroid-, or disuse-induced skeletal muscle atrophy. These analyses demonstrated that activation of VPAC2R, but not VPAC1R, reduced the loss of skeletal muscle mass and force during conditions of skeletal muscle atrophy resulting from corticosteroid administration, denervation, casting-induced disuse, increased skeletal muscle mass, and force of nonatrophying muscles. These studies indicate that VPAC2R agonists may have utility for the treatment of skeletal muscle-wasting diseases.

VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells.

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.

Evidence for multiple rat VPAC1 receptor states with different affinities for agonists.

We compare the binding properties of [125I-VIP] and [125I]-Ro 25 1553 to VPAC1 receptors, expressed in stably transfected CHO cells. [125I]-VIP labelled two VPAC1 receptor states, while [125I]-Ro 25 1553 labelled selectively a limited number of high-affinity receptors. This high-affinity state probably corresponds to an agonist-receptor-Gs ternary complex as its properties (guanyl nucleotides, EC50 values and maximal effect) were affected by cholera toxin pre-treatment. Both high- and low-affinity receptors participated in the adenylate cyclase activation. This suggested that agonists activate not only low-affinity uncoupled receptors by facilitating the ternary complex formation, but also activated the high-affinity ternary complex by accelerating the GTP binding to emptied, receptor-bound G proteins.

Distribution of the VPAC2 receptor in peripheral tissues of the mouse.

The neuropeptide vasoactive intestinal peptide (VIP) exerts its actions through two structurally related G protein-coupled receptors (VPAC(1) and VPAC(2)). Pituitary adenylate cyclase-activating polypeptide (PACAP) is also a potent agonist of VPAC(1) and VPAC(2) receptors as well as of a third, PACAP-specific receptor (PAC(1)). We report here the distribution of the VPAC(2) receptor in peripheral tissues of the mouse, determined by receptor autoradiography using [(125)I]VIP and the selective VPAC(2) receptor agonist [(125)I]Ro25-1553 in wild-type and VPAC(2) receptor-null mice. In addition, displacement experiments with the VPAC(2)-selective agonist Ro25-1553 and the VPAC(1)-selective agonist [K(15),R(16),L(27)]VIP(1-7)/GRF(8-27) were performed using the universal radioligand [(125)I]VIP. The VPAC(2) receptor is found predominantly in smooth muscle (in blood vessels and in the smooth muscle layers of the gastrointestinal and reproductive systems), the basal part of the mucosal epithelium in the colon, lung, the vasculature of the kidney, adrenal medulla, and retina. Unexpectedly, the receptor was also present in thyroid follicular cells and acinar cells of the pancreas, tissues that have not been found to express the receptor in other species, and in very large amounts in the lung. Our data suggest novel functions of the VPAC(2) receptor and additional potential therapeutic uses of drugs acting at the receptor (including the treatment of erectile dysfunction), but our results also indicate that caution should be exercised in using the mouse as an animal model for the evaluation of VIP analogs intended for diagnostic or therapeutic use in man.

VIP and drug design.

The following review outlines the physiological outcome of VIP and VIP gene manipulations. Previously, we reviewed the various VIP receptors associated with biological functions ranging from growth regulation, sexual function, bronchodilation, vasodilation and immune interactions to neurotrophism. VIP-based drug design is discussed below.

Vasoactive intestinal peptide (VIP) stimulates cortisol secretion from the H295 human adrenocortical tumour cell line via VPAC1 receptors.

Vasoactive intestinal peptide (VIP) shows a wide tissue distribution and exerts numerous physiological actions. VIP was shown in a dose-dependent manner to increase cortisol secretion in the NCI-H295R human adrenocortical carcinoma (H295) cell line (threshold dose 3.3x10(-10) M, maximal dose 10(-7) M), coupled with a parallel increase in cAMP accumulation. Receptor-specific agonists were employed to determine which of the two known VIP receptor subtypes was involved in cortisol secretion. Treatment with the VPAC1 receptor agonist, [K(15), R(16), L(27)]VIP(1-7)/GRF(8-27), produced a dose-dependent increase in H295 cell cortisol secretion (threshold dose 10(-11) M, maximal dose 10(-7) M) similar to that seen with VIP. Meanwhile, the high-affinity VPAC2 receptor agonist, RO-25-1553, failed to stimulate significantly cortisol or cAMP production from H295 cells. Inhibition of VIP-mediated H295 cell cortisol secretion by PG97-269, a competitive VPAC1-specific antagonist, produced parallel shifts of the dose-response curve and a Schild regression slope of 0.99, indicating competitive inhibition at a single receptor subtype. VIP is known also to interact with the PAC1 receptor, albeit with lower affinity (EC(50) of approximately 200 nM) than the homologous ligand, PACAP (EC(50) of approximately 0.5 nM). PACAP stimulated cortisol secretion from H295 cells (EC(50) of 0.3 nM), suggesting the presence of functional PAC1 receptors. However, stimulation of cortisol secretion by nanomolar concentrations of VIP (EC(50) of 5 nM), coupled with real-time PCR estimation that VPAC1 receptor transcripts appear 1000-fold more abundant than PAC1 transcripts in H295 cells, makes it unlikely that VIP signals via PAC1 receptors. Together, these data suggest that VIP directly stimulates cortisol secretion from H295 cells via activation of the VPAC1 receptor subtype.

Lysine 195 and aspartate 196 in the first extracellular loop of the VPAC1 receptor are essential for high affinity binding of agonists but not of antagonists.

The role in ligand recognition and receptor activation of two adjacent charged residues (lysine 195 and aspartate 196) in the first extracellular loop of the human VPAC(1) receptor was investigated in stably transfected CHO cells expressing the wild type or point mutated receptors.Replacement of lysine 195 by glutamine or of aspartate 196 by asparagine reduced the agonists' ability to stimulate adenylate cyclase activity; VIP behaved like a partial agonist and a partial agonist behaved as an antagonist. The receptor's capacity to recognize agonists was reduced but antagonists' affinity was unaffected. Both results suggesting that the two charged residues are essential for VPAC(1) receptor activation. On the other hand, the double mutant was less severely affected than single mutants suggesting that hydrogen bonds may partially compensate the loss of charged residues. But the inversion of the residues affected receptor recognition and activation more markedly suggesting that the two charged residues do not interact directly.

The role of VIP/PACAP receptor subtypes in spinal somatosensory processing in rats with an experimental peripheral mononeuropathy.

Peripheral nerve damage often results in the development of chronic pain states, resistant to classical analgesics. Since vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are up-regulated in dorsal root ganglion cells following peripheral nerve injury, we investigated the expression and influence of VPAC1, VPAC2 and PAC1 receptors in rat spinal dorsal horn following a chronic constriction injury (CCI). Electrophysiological studies revealed that selective antagonists of VPAC1, VPAC2 and PAC1 receptors inhibit mustard oil-, but not brush-induced activity of dorsal horn neurones in CCI animals, while cold-induced neuronal activity was attenuated by VPAC1 and PAC1, but not VPAC2 receptor antagonists. Ionophoresis of selective agonists for the receptor subtypes revealed that the VPAC2 receptor agonist excited twice as many cells in CCI compared to normal animals, while the number of cells excited by the VPAC1 receptor agonist decreased and responses to PACAP-38 remained unchanged. In situ hybridisation histochemistry (ISHH) confirmed an increase in the expression of VPAC2 receptor mRNA within the ipsilateral dorsal horn following neuropathy, while VPAC1 receptor mRNA was seen to decrease and that for PAC1 receptors remained unchanged. These data indicate that VIP/PACAP receptors may be important regulatory factors in neuropathic pain states.