PC12 Cell Membranes Research Articles

Synaptotagmin 1-mediated cell membrane penetration and dopamine release enhancement by latroeggtoxin-VI

Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus, was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency.

Study on the Application of a Novel Nano-Curcumin Drug for Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disease hallmarked by impaired cognitive function and memory decline; it is accompanied by a variety of mental symptoms and behavioral disorders. This disease has a serious impact on society, families, and the afflicted patients. Curcumin (Cur) nanoparticles were preparation by modifying curcumin with polyethylene glycol-polylactic acid (PEG-PLA) amphiphilic block copolymer. The resulting nanodrug can successfully penetrate the blood brain barrier (BBB) and concentrate in the main pathological areas of AD. As measured with a Zeta particle size analyzer, the particle size of PEG-PLA/Cur was 100 ± 0.53 nm, and it was electrically neutral. In vitro experiments revealed that PEG-PLA/Cur successfully penetrated the membrane of PC12 cells and inhibited proliferation. PEG-PLA/Cur also exhibited an inhibitory effect on the production of amyloid β protein (Aβ fiber) and depolymerization activity on Aβ fiber aggregates. PEG-PLA/Cur represents a novel nanodrug with the potential to treat AD.

Elucidation of the Mechanisms for the Underlying Depolarization and Reversibility by Photoactive Molecule.

Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.

Attachment of streptavidin-modified superparamagnetic iron oxide nanoparticles to the PC-12 cell membrane

Efficient attachment of magnetic nanoparticles to cell membranes plays an important role in the activation of cell membrane channels. Streptavidin (SA) was successfully modified to Poly (ethylene imine) (PEI)-superparamagnetic iron oxide nanoparticles (SPIONs) to form SA/PEI-SPIONs, which have high colloidal stability and low cytotoxicity. The SA/PEI-SPIONs were incubated with PC-12 cells which had first been cultured in a Roswell Park Memorial Institute medium 1640 containing 0.2 mg l−1 biotin for 12 h. The cells were observed by transmission electron microscopy, and the nanoparticles were clearly attached on the cell membrane, which can be attributed to the specific binding between the SA and biotin sites on the cell surface. This work provides a simple way to attach SA-modified nanoparticles on the membranes of cells by only culturing cells in a biotin-containing medium. This work makes possible biomedical applications that require nanoparticles to target cell membranes.

Relative quantification of deuterated omega-3 and -6 fatty acids and their lipid turnover in PC12 cell membranes using TOF-SIMS

Understanding FA metabolism and lipid synthesis requires a lot of information about which FAs and lipids are formed within the cells. We focused on the use of deuterated substrates of 100 μM α-linolenic acid and linoleic acid to determine the relative amounts of their converted PUFAs and specific phospholipids that are incorporated into cell plasma membranes. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to image and analyze lipids in model cell membranes with and without FA treatment. Because of its high spatial resolution, TOF-SIMS can be used to simultaneously provide both chemical information and distribution of various molecules in the sample surface down to the subcellular scale. Data obtained from this analysis of isotopes in the cell samples were used to calculate the relative amounts of long-chain PUFAs and phospholipids from their precursors, α-linolenic acid and linoleic acid. Our results show that the FA treatments induced an increase in the amounts of α-linolenic acid and linoleic acid and their long-chain conversion products. Moreover, an enhanced level of phospholipid turnover of these FAs in lipids such as phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols was also observed in the cell plasma membrane.

Protective Effect of Quercetin against Oxidative Stress-Induced Cytotoxicity in Rat Pheochromocytoma (PC-12) Cells

Oxidative stress has been implicated in the pathogenesis of many kinds of neurodegenerative disorders, particularly Parkinson’s disease. Quercetin is a bioflavonoid found ubiquitously in fruits and vegetables, and has antioxidative activity. However, the underlying mechanism of the antioxidative effect of quercetin in neurodegenerative diseases has not been well explored. Here, we investigated the antioxidative effect and underlying molecular mechanisms of quercetin on PC-12 cells. We found that PC-12 cells pretreated with quercetin exhibited an increased cell viability and reduced lactate dehydrogenase (LDH) release when exposed to hydrogen peroxide (H2O2). The significantly-alleviated intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and lipoperoxidation of the cell membrane of PC-12 cells induced by H2O2 were observed in the quercetin pretreated group. Furthermore, quercetin pretreatment markedly reduced the apoptosis of PC-12 cells and hippocampal neurons. The inductions of antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in PC-12 cells exposed to H2O2 were significantly reduced by preatment with quercetin. In addition, quercetin pretreatment significantly increased Bcl-2 expression, and reduced Bax, cleaved caspase-3 and p53 expressions. In conclusion, this study demonstrated that quercetin exhibited a protective effect against oxidative stress-induced apoptosis in PC-12 cells. Our findings suggested that quercetin may be developed as a novel therapeutic agent for neurodegenerative diseases induced by oxidative stress.

The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis

Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming.

THE 18 kDa SIGMA‐2 Receptor and PGRMC1 are Derived From Separate Genes

The sigma‐2 receptor (S2R) is a unique binding site expressed in many mammalian tissues. Due to high expression in tumors, the S2R is a promising target for cancer diagnostic imaging. However, the sequence of the S2R has remained a matter of debate. Historically the identity of S2R has been determined by these criteria: (1) the S2R binds with high affinity to 1,3‐di‐o‐tolylguanidine (DTG) and haloperidol but not the selective sigma‐1 receptor ligand, (+)‐pentazocine; (2) specific photolabeling with [3H]Azido‐DTG, and [125I]‐iodoazido‐fenpropimorph ([125I]IAF) has shown the apparent molecular size of S2R to be 18‐21kDa. The progesterone receptor membrane component 1 (PGRMC1), a 25 kDa protein, has been recently reported to be the S2R. To establish the identity of the S2R, we created a PGRMC1 knockout NSC34 cell line using CRISPR/Cas9 technology. We found that in NSC34 cells devoid of PGRMC1, the maximum binding (Bmax) of haloperidol protectable [3H]‐DTG binding was similar to that of wild‐type control cells. Moreover, in both control and PGRMC1‐KO cells, [125I]IAF selectively photolabeled a DTG protectable S2R‐18kDa protein. A series of previously reported highly selective S2R compounds showed protection of [125I]‐IAF photolabeling of the S2R‐18kDa in PC12 cell membranes that contain both PGRMC1 and S2R‐18kDa. These results support the conclusion that PGRMC1 and S2R‐18kDa are separate proteins derived from two different genes. Cloning and characterization of the S2R‐18kDa will provide further clarification.

Spectroscopic investigation of local mechanical impedance of living cells.

We studied nanoscale mechanical properties of PC12 living cells with a Force Feedback Microscope using two experimental approaches. The first one consists in measuring the local mechanical impedance of the cell membrane while simultaneously mapping the cell morphology at constant force. As the interaction force is increased, we observe the appearance of the sub-membrane cytoskeleton. We compare our findings with the outcome of other techniques. The second experimental approach consists in a spectroscopic investigation of the cell while varying the tip indentation into the membrane and consequently the applied force. At variance with conventional dynamic Atomic Force Microscopy techniques, here it is not mandatory to work at the first oscillation eigenmode of the cantilever: the excitation frequency of the tip can be chosen arbitrary leading then to new spectroscopic AFM techniques. We found in this way that the mechanical response of the PC12 cell membrane is found to be frequency dependent in the 1 kHz - 10 kHz range. In particular, we observe that the damping coefficient consistently decreases when the excitation frequency is increased.

Imaging a Single Living Cell via Shear Force-based Scanning Ion Conductance Microscopy in Standing Approach Mode with Differential Control

A single living cell (PC12), cultured on a polystyrene dish and an albumin island immobilized on glass, were successfully imaged utilizing the differential standing approach (d-SA) mode of a scanning ion conductance microscope (SICM). In the d-SA mode, the differential of the output voltage of a tuning fork (rather than its absolute value) is used as the signal for maintaining a constant distance between the probe of a scanning ion conductance microscope (SICM) and the sample. The topography and SICM images were obtained simultaneously, and approach curves, when the tip approached the immobilized albumin or the PC12 cell membrane, were also obtained. The settings for the d-SA mode were carefully determined from the PC12 cell approach curve. Furthermore, differences in the topographies of the immobilized albumin and living PC12 cell images obtained using the standing approach (SA) versus d-SA mode are discussed. It is shown that SICM in d-SA mode is capable of imaging soft samples such as living cells without damaging them.

Direct Measurement of Secretory Vesicle-Plasma Membrane Tethering Interactions by Correlated AFM Force-Clamp and TIRF Microscopy

The molecular mechanisms behind the tethering of secretory vesicles to the plasma membrane are not well understood. To study these mechanisms, we use a combined AFM/TIRF method applying forces to GFP-tagged secretory granules tethered to PC-12 cell membrane sheets. In the experiment shown in the figure, the AFM tip captured a granule, and was then lowered onto the sheet (a-b). Pulling forces were applied, ranging from ∼100pN to ∼700pN (c-e). Extension and rupture events are apparent as transient variations in the pulling force, as well as increases in the scanner z position sensor. Change in sensor position is consistent with changes in vesicle height as observed in the TIRF, based on the calibrated excitation decay constant (c-d). Rapid transients with small amplitude reveal partial tether extensions by 10-20nm, while large transients with slow decay reveal more gradual extensions, exceeding 1μm, that appear to be composed of multiple steps. These observations suggest that the dynamics of vesicle tether extensions are much more complex than previously thought. A large data set of recordings like that in the figure is being analyzed. Supported by NIH grant R21NS072577.View Large Image | View Hi-Res Image | Download PowerPoint Slide

BoNT/A Trafficking with FGFR3 in Neuronal Cell Lines Studied with Cross-Correlation RICS

Botulinum neurotoxin serotype A (BoNT/A) is a versatile protein therapeutic used to treat a variety of neuromuscular and pain disorders. BoNT/A is a 150 kDa protein consisting of three domains, including a 50 kDa receptor binding domain (HC/A). To enter neurons, the HC/A binds a receptor complex composed of proteins and gangliosides. Members of the synaptic vesicle (SV2) glycoprotein family were reported to mediate BoNT/A uptake by facilitating toxin binding and internalization during vesicle recycling in depolarized neurons. Previously, we identified Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity component of BoNT/A receptor complex. The interaction between FGFR3 and recombinant HC/A (rHC/A) was analyzed using Cross Correlation RICS (ccRICS). Differentiated SiMa, PC-12, and SH-SY5Y cells expressing a fluorescent FGFR3-Halo tag chimera were treated with Alexa-Fluor®633-rHC/A. These proteins move together on the membrane of SiMa and PC-12 cells with a diffusion rate of 3.2 μm2/s and after vesicular endocytosis, the rHC/A-FGFR3 complex diffuses at a rate of 1.1 μm2/s. The GccRICS (0, 0) of rHC/A and FGFR3 peaks in SiMa, PC-12, and SH-SY5Y after 2, 6, and 9 hours treatment, respectively. We investigated the trafficking of rHC/A in PC-12 cells with GFP labeled markers for early endosomes and synaptic vesicles. The fluorescent signal from rHC/A cross correlated with early endosomes when the cells were treated in basal or depolarizing media. However, cross-correlation with synaptic vesicles was only observed when the cells were depolarized. These data suggest that BoNT/A can enter neurons via the endosomal and/or synaptic vesicle pathways depending on the cell's polarization state. FGFR3 is part of a high-affinity receptor complex for BoNT/A in neuronal membranes and the FGFR3-BoNT/A interaction plays a role in productive trafficking of the toxin in neuronal cells.

Correlative 3-Dimensional Super-Resolution Fluorescence and Tomographic Electron Microscopy of Endocytic Proteins at the Nanometer Scale

Three-dimensional super-resolution fluorescence microscopy methods such as interferometric photoactivated localization microscopy (iPALM) can localize individual proteins of interest at high resolution (20 nm in XY and 8 nm in Z). These images, however, lack the structural context that could be provided by other imaging methods including electron microscopy. Here, we develop a method that combines iPALM and 3D tomographic transmission electron microscopy (TEM). First, we use iPALM to localize fluorescently-tagged endocytic proteins on the inner plasma membrane of PC12 cells in three dimensions. Next, platinum replicas of these same membranes are imaged with transmission electron microscopy to create tomograms. Finally, both images are combined to create a map of the nanometer scale location of these proteins within their three dimensional cellular context. This technique has the power to build ultra high resolution 3D topographic maps of molecular structures in the context of their native cellular environment.

Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters

Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.

Segregation of PIP2 and PIP3 into distinct nanoscale regions within the plasma membrane

SummaryPIP2 and PIP3 are implicated in a wide variety of cellular signaling pathways at the plasma membrane. We have used STORM imaging to localize clusters of PIP2 and PIP3 to distinct nanoscale regions within the plasma membrane of PC12 cells. With anti-phospholipid antibodies directly conjugated with AlexaFluor 647, we found that PIP2 clusters in membrane domains of 64.5±27.558 nm, while PIP3 clusters had a size of 125.6±22.408 nm. With two color direct STORM imaging we show that >99% of phospholipid clusters have only one or other phospholipid present. These results indicate that lipid nano-domains can be readily identified using super-resolution imaging techniques, and that the lipid composition and size of clusters is tightly regulated.

Utilization of Photoinduced Charge-Separated State of Donor–Acceptor-Linked Molecules for Regulation of Cell Membrane Potential and Ion Transport

The control of ion transport across cell membranes by light is an attractive strategy that allows targeted, fast control of precisely defined events in the biological membrane. Here we report a novel general strategy for the control of membrane potential and ion transport by using charge-separation molecules and light. Delivery of charge-separation molecules to the plasma membrane of PC12 cells by a membranous nanocarrier and subsequent light irradiation led to depolarization of the membrane potential as well as inhibition of the potassium ion flow across the membrane. Photoregulation of the cell membrane potential and ion transport by using charge-separation molecules is highly promising for control of cell functions.

Phosphatidyl-Serine-Positive Cells with High Sensitivity to the Alzheimer's Disease Aβ Peptides Display Distinctive Mitochondrial Characteristics

The Alzheimer's Aβ peptides exhibit cell-selective toxic effects in cultured cells and in neurons of Alzheimer's disease (AD) brains. As we previously showed ex vivo neuronal cells and cell lines consisted of subpopulations of cells characterized by differential levels of a common surface membrane target for the phosphatidyl-serine (PtdSer) specific binder annexin V and for the Alzheimer's Aβ peptidesbinding. These findings enable us to efficiently separate by flow cytometry sorting PtdSer positive cells on a variety of neuronal cell lines and ex vivo neurons and analyze their Aβ-binding affinity. We collected compelling data confirming the involvement of PtdSer as one of the surface membrane signal molecules for Aβ. This investigation used the persistent presence of PtdSer on the outer leaflet of the plasma membrane of PC12 cells to identify, sort and further culture the subpopulation of cells that are PtdSer positive (PtdSer+) and have high affinity to bind Aβ, and consequently are more likely to be harmed by Aβ. Various viability tests showed that the group of cells sorted as PtdSer+ was the most sensitive to Aβ toxic effects. The caspase3/7 activation level of the PtdSer+ subpopulation of cells was similar to one of the PtdSer negative (PtdSer-) subpopulation and the control cells. Therefore we assume, PtdSer+ cells are not undergoing apoptosis. Additionally, PtdSer+ cells have persistent higher levels of PtdSer on the outer leaflet of the plasma membrane, significantly lower levels of cytosolic ATP, lower mitochondrial mass and mitochondrial membrane potential, and slightly higher production of reactive oxidative species compared to PtdSer- and control cells. We hypothesize that all of these distinctive cellular and mitochondrial conditions are the basis for the high sensitivity to Aβ displayed by some selective cell lines and neurons.

Effect of “Chemical” Hypoxia on the Potassium Conductance of the Membrane of Pheochromocytoma Cells

We studied the effect of “chemical” (induced by the action of sodium thiosulfate, STS) hypoxia on the potassium conductance of the membrane of pheochromocytoma cells. Application of 1 to 10 mM STS decreased in a dose-dependent manner the amplitude of integral potassium current without changes in the voltage dependence of its activation. The concentration dependence of the action of STS on the amplitude of potassium current was estimated using the Boltzmann equation. The value of concentration for 50% inhibition was 2.7 ± 0.2 mM, while the slope coefficient was 0.9 ± 0.2 mM−1. In the presence of 10 mM STS, the decrease in the amplitude of potassium current reached, on average, 55%. Therefore, “chemical” hypoxia influences rather significantly the potassium conductance of the membrane of pheochromocytoma PC12 cells.

Membrane protein sequestering by ionic protein–lipid interactions

Neuronal exocytosis is catalyzed by the SNARE protein syntaxin-1A1. Syntaxin-1A is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis2,3. However, how syntaxin-1A is sequestered is unknown. Here, we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We found with super-resolution STED microscopy on the plasma membrane of PC12 cells that PIP2 is the dominant inner-leaflet lipid in ~73 nm-sized microdomains. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the C-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases.

CART peptide stimulation of G protein-mediated signaling in differentiated PC12 Cells: Identification of PACAP 6–38 as a CART receptor antagonist

CART peptides are peptide neurotransmitters and hormones that are involved in many different physiological responses. While much is known about the peptides regarding their structure, processing and gene regulation, less is known about their postsynaptic actions and receptors. Using (125)I-CART 61-102 as a ligand and unlabeled CART 61-102 or CART 55-102 as displacers, high-affinity specific binding was detected in PC12 cells. Differentiation of the PC12 cells increased specific binding several-fold. The increase in specific binding found after differentiation was inhibited by actinomycin D and cycloheximide, suggesting that the increase in specific binding was dependent on RNA and protein synthesis. CART 1-27, a peptide that has never been shown to elicit responses, did not displace (125)I-CART 61-102 binding, nor did more than 20 other peptides that were examined. Surprisingly, however, PACAP 1-38 and PACAP 6-38 were found to be low-affinity inhibitors of CART binding. CART treatment increased binding of (35)S-GTPgamma-S to PC12 cell membranes. Moreover, CART treatment of intact PC12 cells elicited robust increases in phospho-ERK in a manner that was increased with differentiation, blocked by pertussis toxin and antagonized by PACAP 6-38. These findings extend previous research and suggest that the CART binding site in PC12 cells reflects a G protein-coupled receptor linked with Gi/o, and also demonstrate that PACAP 6-38 may be useful as a CART receptor antagonist.