Tonic activation of VPAC1 receptors by VIP modulates theta-burst induced LTP in the hippocampus: transduction pathways and GABAergic mechanisms.

Background and purpose Vasoactive intestinal peptide (VIP), acting on both VPAC and VPAC receptors, is a key modulator of hippocampal synaptic transmission, pyramidal cell excitability and synaptic plasticity phenomena, like long-term depression (LTD), partly through modulation GABAergic disinhibitory circuits. VIP effects on LTP and the involvement of disinhibition were scarcely investigated. Experimental approach The influence of endogenous VIP on CA1 LTP induced by TBS was evaluated in the CA1 area of hippocampal slices using field-excitatory electrophysiological recordings from young-adult Wistar rats using selective VPAC and VPAC antagonists. Phosphorylation of GluA1 AMPA receptor subunits and Kv4.2 potassium channels was evaluated in hippocampal membranes obtained from such slices by Western blot. Key results Here we show that VIP, acting on VPAC (but not VPAC) receptors, is an endogenous inhibitor of hippocampal LTP induced by theta-burst stimulation (TBS) in the CA1 area of the hippocampus of young adult Wistar rats. This effect is dependent on GABAergic transmission and relies on the integrity of NMDA and CaMKII-dependent LTP expression mechanisms but not on PKA and PKC activity. Furthermore, it regulates the expression and Serphosphorylation of Kv4.2 potassium channels responsible for the A-current while inhibiting phosphorylation of Kv4.2 on Thr. Conclusions and implications Altogether this suggests that endogenous VIP controls the expression of hippocampal CA1 LTP by regulating disinhibition through activation of VPAC receptors in interneurons. This may impact the expression and phosphorylation of Kv4.2 K channels at hippocampal pyramidal cell dendrites.

Comparative Study of Senescent Th Biomarkers in Healthy Donors and Early Arthritis Patients. Analysis of VPAC Receptors and Their Influence

Pro-inflammatory CD4+CD28− T cells are characteristic of immunosenescence, but also of several autoimmune/inflammatory diseases. Vasoactive intestinal peptide (VIP) acts as an anti-inflammatory and immunomodulatory mediator on these cells. Our objective was to study the mutual influence between senescent Th cells and VIP axis in early arthritis (EA), comparing with non-EA donors. We characterized the correlation between senescent Th cells and clinic parameters of EA as well as the behavior of senescent Th biomarkers by real-time PCR. Clinical data were systematically recorded at baseline and after 6 months of follow-up. The number of CD4+CD28− T cells measured by sorting is higher in patients who initially meet ACR classification criteria for rheumatoid arthritis (RA) compared to those who were classified as undifferentiated arthritis (UA). A slight positive correlation between EA CD4+CD28− T cells and CRP or ESR and a negative correlation with bone mineral density were found. Th senescent biomarkers in EA CD4+CD28− T cells were similar to donors, however some of them increased after 6 months of follow-up. VPAC receptors were analyzed by real-time PCR and immunofluorescence, and CD4+CD28− T cells showed higher expression of VPAC2 and lower of VPAC1, VPAC2 showing a significant increased expression in EA cells. Sorted CD4+CD28− T cells were in vitro expanded in presence of VIP, wherein VIP increased senescent biomarker CD27, while it diminished CD57 or NKG2 senescent biomarkers. Our study demonstrates for the first time the existence of a link between senescent Th cells and the VIP axis.

PET imaging urothelial bladder cancer: A novel approach.

443 Background: Urothelial bladder cancer (UBC) inflicts >80,000 new patients annually. Since treatment is stage-dependent, accurate staging is crucial. Conventional imaging and biopsy are often unreliable. A large number of PET tracers, developed to improve imaging, have limitations e.g. urinary excretion compromising their ability to assess the bladder lumen and invasive tumors. This study is to validate a hypothesis that high density VPAC receptors expression on UBC cell surface, can be targeted to PET image UBC, to determine loco-regional disease and metastatic lesions. Methods: Cu-64-TP3805 (4±10% mCi), with its high affinity (3.1 x 10−9M) for VPAC, was given IV to 19 UBC patients (44-80 yrs), scheduled for radical cystectomy. Those eligible for neoadjuvant chemotherapy were treated as such. Urine and blood samples were collected on the day of scan. Whole body PET/CT images acquired 60 to 90 min later and read by two physicians. Surgery was performed 1 to 4 weeks later. Imaging results were correlated with histology. Results: There were no adverse events. Urinary excretion of Cu-64-TP3805 was negligible. Blood clearance was biphasic (t ½ a = 22.3 ±2.7 min ~ 85% and t ½ β = 118.2 ± 4.9 min ~ 15%). VPAC PET bladder images were true positive (TP) in 11, true negative (TN) in 4, false positive (FP) in 1 and false negative (FN) in 3 patients with 79% sensitivity (95% CI 49%-95%), 80% specificity (95% CI 28%-100%), 92% PPV (95% CI 62%-100%), and 57% NPV (95% CI 18%-90%). Prostate images were TP in 8, TN in 6, and FP in 5 patients, with 100% sensitivity (95% CI 63%-100%), 55% specificity (95% CI 23%-83%), 62% PPV (95% CI 32%-86%), and 100% NPV (95% CI 54%-100%). The 5 FP images revealed HGPIN on re-analysis. For lymph nodes, images were TP in 1, TN in 14 and FN in 4 patients, with 25% sensitivity (95% CI 1%-81%), 100% specificity (95% CI 78%-100%), 100% PPV (95% CI 3%-100%), and 83% NPV (95% CI 59%-96%). In one patient, several lesions were seen in the spine and iliac crest. Biopsy was positive for metastasis. In smokers (N=12) there was diffused or focal tracer uptake in the lungs. In 7 non-smokers, 3 with CT depicted abnormality had tracer lung uptake and 4 did not. Conclusions: These first in human pilot study data depict Cu-64-TP3805 VPAC targeting to image UBC as worthy of further investigation.

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20–70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6–38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, “hop” splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and α-helical regions of PACAP-27 are required for initiating gallbladder contraction.

Mutual dependence of VIP/PACAP and CCK receptor signaling for a physiological role in duck exocrine pancreatic secretion

Unlike in rodents, CCK has not been established as a physiological regulator in avian exocrine pancreatic secretion. In the isolated duck pancreatic acini, 1 nM CCK was required for stimulation of amylase secretion, maximal effect being achieved at 10 nM; picomolar CCK was without effect. Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 (10-12-10-7 M) alone had no effect, but made picomolar CCK effective. VPAC agonist VIP 10-10-10-7 M stimulated amylase secretion marginally, but made CCK 10-12-10-10 M effective also. PACAP-27 and VIP both shifted the maximal CCK concentration from 10-8 to 10-9 M. This sensitizing effect was mimicked by forskolin. CCK dose dependently induced intracellular Ca2+ concentration ([Ca2+]i) oscillations. PACAP-38 (1 nM), PACAP-27 (1 nM), VIP (10 nM), or forskolin (10 μM) alone did not stimulate [Ca2+]i increase, neither did they modulate CCK (1 nM)-induced oscillations; but when they were added to cells simultaneously exposed to subthreshold CCK (10 pM), calcium spikes emerged. Amylase secretion induced by the simultaneous presence of 10 pM CCK and VPAC agonists was completely blocked by removing extracellular calcium, but the protein kinase C inhibitor staurosporine (1 μM) was without effect. CCK (10 nM)-induced secretion was inhibited by CCK1 receptor antagonist FK480 (1 μM). Gastrin from 10-12 to 10-6 M did not stimulate amylase secretion nor did it (100 nM) induce [Ca2+]i increase. The above data suggest that duck pancreatic acini possess both CCK1 and VPAC receptors; simultaneous activation of both is required for each to play a physiological role.