Process Development of a Macrocyclic Peptide Inhibitor of PD-L1.

This article outlines the process development leading to the manufacture of 800 g of BMS-986189, a macrocyclic peptide active pharmaceutical ingredient. Multiple N-methylated unnatural amino acids posed challenges to manufacturing due to the lability of the peptide to cleavage during global side chain deprotection and precipitation steps. These issues were exacerbated upon scale-up, resulting in severe yield loss and necessitating careful impurity identification, understanding the root cause of impurity formation, and process optimization to deliver a scalable synthesis. A systematic study of macrocyclization with its dependence on concentration and pH is presented. In addition, a side chain protected peptide synthesis is discussed where the macrocyclic protected peptide is extremely labile to hydrolysis. A computational study explains the root cause of the increased lability of macrocyclic peptide over linear peptide to hydrolysis. A process solution involving the use of labile protecting groups is discussed. Overall, the article highlights the advancements achieved to enable scalable synthesis of an unusually labile macrocyclic peptide by solid-phase peptide synthesis. The sustainability metric indicates the final preparative chromatography drives a significant fraction of a high process mass intensity (PMI).

Pituitary adenylate cyclase-activating polypeptide-induced PAC1 receptor internalization and recruitment of MEK/ERK signaling enhance excitability of dentate gyrus granule cells.

Pituitary adenylate cyclase activating polypeptide (PACAP; ADCYAP1) is a pleiotropic neuropeptide widely distributed in both the peripheral and central nervous systems. PACAP and its specific cognate PAC1 receptor (ADCYAP1R1) play critical roles in the homeostatic maintenance of multiple physiological and behavioral systems. Notably, maladaptations in the PACAPergic system have been associated with several psychopathologies related to fear and anxiety. PAC1 receptor transcripts are highly expressed in granule cells of the dentate gyrus (DG). Here, we examined the direct effects of PACAP on DG granule cells in brain slices using whole cell patch recordings in current clamp mode. PACAP significantly increased the intrinsic excitability of DG granule cells via PAC1 receptor activation. This increased excitability was not mediated by adenylyl cyclase/cAMP or phospholipase C/PKC activation, but instead via activation of an extracellular signal-regulated kinase (ERK) signaling pathway initiated through PAC1 receptor endocytosis/endosomal signaling. PACAP failed to increase excitability in DG granule cells pretreated with the persistent sodium current blocker riluzole, suggesting that the observed PACAP effects required this component of the inward sodium current.

Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability.

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels (Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

G Protein-Coupled Receptor Endosomal Signaling and Regulation of Neuronal Excitability and Stress Responses: Signaling Options and Lessons From the PAC1 Receptor.

Our understanding of G protein coupled receptor (GPCR) mechanisms and functions have evolved considerably. Among the many conceptual realignments, GPCRs can exist in an ensemble of active microstates that have the potential to differentially engage specific downstream signaling events. Furthermore, among GPCR dynamics, GPCR internalization and vesicular trafficking are no longer solely mechanisms for desensitization, but now appreciated to form intricate endosomal signaling complexes that can potentially target second messengers to intracellular compartments with high temporal and spatial resolution. The PACAPergic system is important in the maintenance of physiological homeostasis in the central and peripheral nervous systems and activation of the PACAP-selective PAC1 receptor can generate differential but coordinate plasma membrane and endosomal signals for cellular responses. The integration of these signals can modulate PACAP-induced changes in ionic conductances that gate neuronal excitability. PACAP/PAC1 receptor generation of endosomal ERK signals participate in chronic pain and anxiety-like responses which can be attenuated with endocytosis inhibitors. From the abilities of ligands to stabilize the different GPCR microstates for biased downstream signaling, the development of biased PAC1 receptor agonists and antagonists may provide opportunities to dissociate the homeostatic regulatory signals of PACAP from the maladaptive effects. In particular, the development of biased antagonists to PAC1 receptor-mediated endosomal signaling may offer therapeutic options for chronic pain and stress-related disorders. J. Cell. Physiol. 232: 698-706, 2017. © 2016 Wiley Periodicals, Inc.

Activation of MEK/ERK signaling contributes to the PACAP-induced increase in guinea pig cardiac neuron excitability.

Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides (Adcyap1) signaling at the selective PAC1 receptor (Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels (Ih) and another flowing through low-voltage-activated calcium channels (IT), which support the peptide-induced increase in excitability. Thus a PACAP- and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.

Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Cav3.1, Cav3.2, and Cav3.3 T-type isoforms and R-type Cav2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization-induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents (Ih). The T-type calcium channel inhibitors, nickel (Ni(2+)) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2-4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni(2+), a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni(2+) also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni(2+)-sensitive current contributes to PACAP-induced modulation of neuronal excitability.

Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (Adcyap1r1) is a G protein-coupled receptor (GPCR) that activates adenylyl cyclase and PLC. Similar to many other GPCRs, our previous studies showed that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a human embryonic kidney (HEK 293) PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization, as determined by treatment with pharmacological inhibitors of endocytosis or temperature reduction, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell-permeable cAMP analogs 8-bromo-cAMP and dibutyryl cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms, in addition to PAC1 internalization/endosome activation, were involved. Inhibition of PAC1 receptor-stimulated PLC/diacylglycerol/PKC signaling by bisindoylmaleimide I also attenuated ERK phosphorylation, and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. Inhibition of PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/diacylglycerol/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation.

Application of fucosylation inhibitors for production of afucosylated antibody.

Fucosylation is an important quality attribute for therapeutic antibodies. Afucosylated antibodies exhibit higher therapeutic efficacies than their fucosylated counterparts through antibody-dependent cellular cytotoxicity (ADCC) mechanism. Since higher potency is beneficial in reducing dose or duration of the treatment, afucosylated antibodies have attracted a great deal of interest in biotherapeutics development. In this study, novel small molecules GDP-D-Rhamnose and its derivatives (Ac-GDP-D-Rhamnose and rhamnose sodium phosphate) were synthesized to inhibit the enzyme in the GDP-fucose synthesis pathway. Addition of these compounds into cell culture increased antibody afucosylation levels in a dose-dependent manner and had no significant impact on other protein quality attributes. A novel and effective mechanism to generate afucosylated antibody is demonstrated for biologics discovery, analytical method development, process development, and other applications.

Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture.

Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, ß4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, ß4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, ß4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.

Synaptic transmission at parasympathetic neurons of the major pelvic ganglion from normal and diabetic male mice.

Bladder and erectile dysfunction are common urologic complications of diabetes and are associated with reduced parasympathetic autonomic control. To determine whether disruption of ganglionic neurotransmission contributes to the loss of function, we investigated synaptic transmission at parasympathetic, major pelvic ganglion (MPG) neurons in control and chronically (20 wk) diabetic mice. In contrast to what has been reported for sympathetic neurons, diabetes did not cause an interruption of synaptic transmission at parasympathetic MPG neurons from streptozotocin-treated C57BL/6J (STZ) or db/db mice. Cholinergically mediated excitatory postsynaptic potentials (EPSPs) were suprathreshold during 5-s trains of 5-, 10-, and 20-Hz stimuli. Asynchronous neurotransmitter release, observed as miniature EPSPs (mEPSPs) during and after stimulation, permitted quantitative assessment of postganglionic, cholinergic receptor sensitivity. mEPSP amplitude following tetanic stimulation (recorded at -60 mV) was reduced in STZ (4.95 ± 0.4 vs. 3.71 ± 0.3 mV, P = 0.03), but not db/db mice. The number of posttetanic mEPSPs was significantly greater in db/db mice at all frequencies tested. Assessment of basic electrophysiological properties revealed that parasympathetic MPG neurons from db/db mice had less negative membrane potentials, lower input resistances, and shorter afterhyperpolarizations relative to their control. MPG neurons from STZ had longer afterhyperpolarizations but were otherwise similar to controls. Membrane excitability, measured by the membrane responsiveness to long-duration (1 s), suprathreshold depolarizing pulses, was unchanged in either model. The present study indicates that, while parasympathetic neurotransmission at the MPG is intact in chronically diabetic mice, obese, type 2 diabetic animals exhibit an altered presynaptic regulation of neurotransmitter release.

The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: implications for neural control of cardiac excitability.

The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10 Hz, 2 min, in the presence of 10 µM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p<0.05) reduction in the magnitude of vagal bradycardia (5 Hz) maintained over the subsequent 20 min (n=6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3 days of tissue culture however, most stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n=6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10 min (1 µM, n=5), and completely reversed with the NPY Y(2) receptor antagonist BIIE 0246 at all time points (1 µM, n=6). Exogenous galanin (n=6, 50-500 nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n=6). Galanin (500 nM) also significantly attenuated the release of (3)H-acetylcholine from isolated atria during field stimulation (5 Hz, n=5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist M40 (n=5). Importantly the GalR(1) receptor was immunofluorescently co-localised with choline acetyl-transferase containing neurons at the sinoatrial node. The protein kinase C inhibitor calphostin (100 nM, n=6) abolished the effect of galanin on vagal bradycardia whilst the protein kinase A inhibitor H89 (500 nM, n=6) had no effect. These results demonstrate that prolonged sympathetic activation releases the slowly diffusing adrenergic co-transmitter galanin in addition to NPY, and that this contributes to the attenuation in vagal bradycardia via a reduction in acetylcholine release. This effect is mediated by GalR(1) receptors on vagal neurons coupled to protein kinase C dependent signalling pathways. The role of galanin may become more important following an acute injury response where galanin expression is increased.

GPCR Intracellular Loop Regulation of Beta-Arrestin-Mediated Endosomal Signaling Dynamics.

G protein-coupled receptors (GPCRs) are currently appreciated to be routed to diverse cellular platforms to generate both G protein-dependent and -independent signals. The latter has been best studied with respect to ß-arrestin-associated receptor internalization and trafficking to signaling endosomes for extracellular signal-regulated kinase (ERK) activation. However, how GPCR structural and conformational variants regulate endosomal ERK signaling dynamics, which can be central in neural development, plasticity, and disease processes, is not well understood. Among class B GPCRs, the PACAP-selective PAC1 receptor is unique in the expression of variants that can contain intracellular loop 3 (ICL3) cassette inserts. The nervous system expresses preferentially the PAC1Null (no insert) and PAC1Hop (28-amino acid Hop insert) receptor variants. Our molecular modeling and signaling studies revealed that the PAC1Null and PAC1Hop receptor variants can associate with ß-arrestin differentially, resulting in enhanced receptor internalization and ERK activation for the PAC1Hop variant. The study amplifies our understandings of GPCR intracellular loop structure/function relationships with the first example of how the duration of endosomal ERK activation can be guided by ICL3. The results provide a framework for how changes in GPCR variant expression can impact developmental and homeostatic processes and may be contributory to maladaptive neuroplasticity underlying chronic pain and stress-related disorders.

PAC1 Receptor Internalization and Endosomal MEK/ERK Activation Is Essential for PACAP-Mediated Neuronal Excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) can significantly increase the excitability of diverse neurons through differential mechanisms. For guinea pig cardiac neurons, the modulation of excitability can be mediated in part by PAC1 receptor plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. By contrast, PAC1 receptor-mediated excitability of hippocampal dentate gyrus granule cells appears independent of membrane-delimited AC/cAMP/PKA and PLC/PKC signaling. For both neuronal types, there is mechanistic convergence demonstrating that endosomal PAC1 receptor signaling has prominent roles. In these models, neuronal exposure to Pitstop2 to inhibit ß-arrestin/clathrin-mediated PAC1 receptor internalization eliminates PACAP modulation of excitability. ß-arrestin is a scaffold for a number of effectors especially MEK/ERK and notably, paradigms that inhibit PAC1 receptor endosome formation and ERK signaling also blunt the PACAP-induced increase in excitability. Detailed PAC1 receptor internalization and endosomal ERK signaling mechanisms have been confirmed in HEK PAC1R-EGFP cells and shown to be long lasting which appear to recapitulate the sustained electrophysiological responses. Thus, PAC1 receptor internalization/endosomal recruitment efficiently and efficaciously activates MEK/ERK signaling and appears to represent a singular and critical common denominator in regulating neuronal excitability by PACAP.

Somatic ATP release from guinea pig sympathetic neurons does not require calcium-induced calcium release from internal stores.

Prior studies indicated that a Ca(2+)-dependent release of ATP can be initiated from the soma of sympathetic neurons dissociated from guinea pig stellate ganglia. Previous studies also indicated that Ca(2+)-induced Ca(2+) release (CICR) can modulate membrane excitability in these same neurons. As Ca(2+) release from internal stores is thought to support somatodendritic transmitter release in other neurons, the present study investigated whether CICR is essential for somatic ATP release from dissociated sympathetic neurons. Caffeine increased intracellular Ca(2+) and activated two inward currents: a slow inward current (SIC) in 85% of cells, and multiple faster inward currents [asynchronous transient inward currents (ASTICs)] in 40% of cells voltage-clamped to negative potentials. Caffeine evoked both currents when cells were bathed in a Ca(2+)-deficient solution, indicating that both were initiated by Ca(2+) release from ryanodine-sensitive stores in the endoplasmic reticulum. Sodium influx contributed to generation of both SICs and ASTICs, but only ASTICs were inhibited by the presence of the P2X receptor blocker PPADs. Thus ASTICs, but not SICs, resulted from an ATP activation of P2X receptors. Ionomycin induced ASTICs in a Ca(2+)-containing solution, but not when it was applied in a Ca(2+)-deficient solution, demonstrating the key requirement for external Ca(2+) in initiating ASTICs by ionomycin. Pretreatment with drugs to deplete the internal stores of Ca(2+) did not block the ability of ionomycin or long depolarizing voltage steps to initiate ASTICs. Although a caffeine-induced release of Ca(2+) from internal stores can elicit both SICs and ASTICs in dissociated sympathetic neurons, CICR is not required for the somatic release of ATP.

The Role of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Signaling in the Hippocampal Dentate Gyrus.

Pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) dysregulation has been associated with multiple stress-related psychopathologies that may be related to altered hippocampal function. In coherence, PACAP- and PAC1 receptor (ADCYAP1R1)-null mice demonstrate changes in hippocampal-dependent behavioral responses, implicating the PACAPergic system function in this structure. Within the hippocampus, the dentate gyrus (DG) may play an important role in discerning the differences between similar contexts, and DG granule cells appear to both highly express PAC1 receptors and receive inputs from PACAP-expressing terminals. Here, we review the evidence from our laboratories and others that PACAP is an important regulator of activity within hippocampal circuits, particularly within the DG. These data are consistent with an increasing literature implicating PACAP circuits in stress-related pathologies such as post-traumatic stress disorder (PTSD) and implicate the hippocampus, and in particular the DG, as a critical site in which PACAP dysregulation can alter stress-related behaviors.

Differential activation of guinea pig intrinsic cardiac neurons by the PAC1 agonists maxadilan and pituitary adenylate cyclase-activating polypeptide 27 (PACAP27).

Pituitary adenylate cyclase-activating polypeptide (PACAP) evokes tachycardia followed by a larger cholinergic bradycardia in isolated guinea pig hearts. We used the selective PAC1 receptor agonist maxadilan and vasoactive intestinal polypeptide (VIP) to test the hypothesis that PACAP27-evoked tachycardia and bradycardia are mediated by VPAC and PAC1 receptors, respectively. Chronotropic actions of these peptides were evaluated in isolated perfused hearts. Direct neuronal actions were determined by intracellular voltage recordings from cholinergic neurons in atrial ganglion whole mounts. Administration of 1 nmol of PACAP27 to isolated hearts evoked typical biphasic rate responses, whereas 1 nmol of maxadilan caused only a minor rate decrease. Desensitization with VIP eliminated the positive chronotropic effect of PACAP27 selectively. Local application of PACAP27 to cardiac neurons frequently evoked slow depolarization and caused prolonged increase of neuronal excitability. Maxadilan rarely affected membrane potential but consistently increased excitability. VIP had no effect on excitability and evoked depolarization in only a few neurons. Because maxadilan increased neuronal excitability but did not trigger action potentials as PACAP often does, we evaluated the interaction of maxadilan with substance P (SP) in isolated hearts. SP depolarizes cardiac neurons more consistently than PACAP, often triggers neuronal action potentials, and causes bradycardia but does not increase neuronal excitability. Maxadilan had a persistent effect to augment negative chronotropic responses to SP. These findings support our hypothesis that PACAP evokes tachycardia and bradycardia through VPAC and PAC1 receptors, respectively. They also suggest that maxadilan and PACAP27 differ in activating PAC1 receptors on cardiac neurons and/or stimulating downstream signaling mechanisms.

Enhancement of Ih, but not inhibition of IM, is a key mechanism underlying the PACAP-induced increase in excitability of guinea pig intrinsic cardiac neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated by PACAP-selective PAC(1) receptors. In dissociated guinea pig cardiac neurons, PACAP causes a positive shift of the voltage dependence of activation of the hyperpolarization-activated nonselective cation current (I(h)). This observation suggested that an enhancement of I(h) contributed to the increase in excitability in neurons within whole-mount cardiac ganglia preparations. To evaluate the role of I(h) in the PACAP-induced increase in excitability, we compared the increase in action potentials generated by 10 nM PACAP in control neurons and in neurons treated with ZD7288 (10 or 100 muM) or CsCl (2 or 2.5 mM), drugs known to inhibit I(h). In control cells exposed to PACAP, 1-s depolarizing current pulses elicited multiple action potential firing in 79% of the neurons. In ZD7288- or CsCl-containing solutions, the 10 nM PACAP-induced increase in excitability was markedly suppressed, with 7% and 21% of the neurons generating multiple action potentials, respectively. Prior results indicated that PACAP initiates depolarization by activating an inward current, which is separate from its enhancement of I(h). Here, we show that a PACAP-induced depolarization was comparable in control neurons and neurons bathed in a CsCl-containing solution, an observation indicating that CsCl did not interfere with activation of the PAC(1) receptor by PACAP. Additional experiments indicated that pretreatment with the putative M current (I(M)) inhibitor 1 mM BaCl(2), but not 10 microM XE991, initiated multiple firing in a majority of neurons, with resting potentials maintained at approximately -60 mV. Furthermore, in Ba(2+)-treated cells, 10 nM PACAP increased the number of action potentials generated. Our results indicate that PACAP enhancement of I(h), rather than inhibition of I(M) and other 1 mM Ba(2+)-sensitive K(+) currents, is a key ionic mechanism contributing to the peptide-induced increase in excitability for neurons within whole-mount cardiac ganglia preparations.

Process research and development for a tetrazole-based growth hormone secretagogue (GHS) pharmaceutical development candidate.

BMS-317180 (1) is a potent, orally active agonist of the human growth hormone secretagogue (GHS) receptor. This manuscript details the process research and development efforts that enabled the synthesis of the phosphate salt of 1 on a multi-kilogram scale. Key considerations in the development of this process focused on safe execution and the requirement for telescoped synthetic transformations (i.e., without isolation of intermediate products) to contend with a lack of suitably crystalline products.