Cardiac output is regulated by the coordinate interactions of stimulatory sympathetic and inhibitory parasympathetic signals. Intracardiac parasympathetic ganglia are integrative centers of cardiac regulation, and modulation of the parasympathetic drive on the heart is accomplished by altering intrinsic cardiac ganglion neuron excitability. The pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) family of peptides modulates cardiac function, and in guinea pig heart, PACAP appears to act directly on intrinsic parasympathetic cardiac ganglia neurons through PACAP-selective receptors. A multidisciplinary project tested whether cardiac PACAP peptides act through PACAP-selective receptors as excitatory neuromodulators amplifying the parasympathetic inhibition from guinea pig cardiac ganglia. The in vivo sources of regulatory PACAP peptides were localized immunocytochemically to neuronal fibers and a subpopulation of intrinsic postganglionic cardiac neurons. RT-PCR confirmed that cardiac ganglia expressed proPACAP transcripts and have PACAP peptide biosynthetic capabilities. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in cardiac ganglia. Alternative splicing of PAC1 receptor transcripts produced predominant expression of the very short variant with neither HIP nor HOP cassettes; lower levels of the PAC1HOP2 receptor mRNA were present. Almost all of the parasympathetic neurons expressed membrane-associated PAC1 receptor proteins, localized immunocytochemically, which correlated with the population of cells that responded physiologically to PACAP peptides. PACAP depolarized cardiac ganglia neurons and increased neuronal membrane excitability. The rank order of peptide potency on membrane excitability in response to depolarizing currents was PACAP27>PACAP38>VIP. The PACAP-induced increase in excitability was not a function of membrane depolarization nor was it caused by alterations in action potential configuration. These results support roles for PACAP peptides as integrative modulators amplifying, through PACAP-selective receptors, the parasympathetic cardiac ganglia inhibition of cardiac output.

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Following G protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex often is rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. Formation of signaling endosomes is recognized to be a mechanism to produce sustained intracellular signals, which 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 (Adcyap1r1). In the current studies, we examined whether PAC1 receptor endocytosis participates in regulation of neuronal excitability. While PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynaminI/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 and dynasore and low temperature to suppress PAC1 receptor endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1 receptor signaling still increased neuronal cAMP production even with endocytic blockade. In aggregate, these studies demonstrate that PACAP/PAC1 receptor complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

A highly enantioselective and practical synthesis of the HIV-1 reverse transcriptase inhibitor efavirenz (1) is described. The synthesis proceeds in 62% overall yield in seven steps from 4-chloroaniline (6) to give efavirenz (1) in excellent chemical and optical purity. A novel, enantioselective addition of Li-cyclopropyl acetylide (4a) to p-methoxybenzyl-protected ketoaniline 3a mediated by (1R,2S)-N-pyrrolidinylnorephedrine lithium alkoxide (5a) establishes the stereogenic center in the target with a remarkable level of stereocontrol.

Recent reports indicate that a considerable amount of heterogeneity exists amongst cardiac postganglionic neurons in their chemical coding patterns and electrical properties, and that some of these cells may serve in roles as sensory and interganglionic neurons as well as motor neurons. This study was undertaken to ascertain whether or not all of these neurons are cholinergic by immunostaining whole-mount preparations of the guinea pig heart for choline acetyltransferase (ChAT). Counts of neurons that were immunostained for microtubule-associated protein-2 revealed that about 1000 neurons exist in about 100 ganglia on the posterior atrial surface. ChAT immunoreactivity was expressed by all of the postganglionic neurons in the cardiac ganglia, including the 5% of neurons that also expressed immunoreactivity for nitric oxide synthase. Varicose nerve fibers that were immunoreactive for ChAT were abundant in ganglia, with every cardiac neuron lying in close apposition to one or more labelled varicosities. ChAT-immunoreactive nerve fibers were also observed in large vagosympathetic fiber bundles, in interganglionic fiber bundles, and passing individually within the myocardium. Immunoreactivity for ChAT was also observed in a large proportion of the small tyrosine hydroxylase-immunoreactive neurons that exist in guinea pig cardiac ganglia. These results indicate that all postganglionic neurons in guinea pig cardiac ganglia are likely to utilize acetylcholine as a neurotransmitter, regardless of their functional role in circuitry of cardiac innervation, and each of these neurons is likely to receive cholinergic input.