Cholinesterases in cardiac ganglia and modulation of canine intrinsic cardiac neuronal activity.

Cholinergic neurotransmission plays a significant role in intrinsic cardiac ganglia with the action of acetylcholine being terminated by acetylcholinesterase (AChE, EC 3.1.1.7). Anatomical studies were performed to characterize neurons associated with AChE and a closely related enzyme, butyrylcholinesterase (BuChE, EC 3.1.1.8), in canine intrinsic cardiac ganglia. Histochemical staining for AChE and BuChE in canine right atrial neurons showed that there were four neuronal populations, namely, those that contained AChE only, BuChE only, both AChE and BuChE, and those that did not contain either enzymes. The neuronal activity of intrinsic cardiac neurons in response to substrates and inhibitors of cholinesterases were studied in anesthetized dogs. The activity of intrinsic cardiac neurons, as measured by changes in the number of action potentials, increased by local application of acetylcholine. However, local application of butyrylcholine led to a considerably greater increase in the activity of intrinsic cardiac neurons. In keeping with the neurochemical heterogeneity in intrinsic cardiac ganglia with respect to cholinesterases, the activity generated by most butyrylcholine-sensitive neurons was not influenced by acetylcholine and the activity generated by the most acetylcholine-sensitive neurons was not influenced by butyrylcholine. This suggests that these two agents preferentially influence different populations of intrinsic cardiac neurons. Enzyme kinetic studies demonstrated that canine AChE preferentially catalyzed the hydrolysis of acetylcholine while canine BuChE preferentially catalyzed the hydrolysis of butyrylcholine. Cholinesterase inhibitors Ro 2-1250 and Ro 2-0638 inhibited both canine cholinesterases, while huperzine A preferentially inhibited canine AChE and ethopropazine inhibited canine BuChE. The activity of neurons in the intrinsic cardiac ganglia significantly increased when Ro 2-1250 or Ro 2-0638 was administered locally. The activity of neurons was not affected when huperzine A or ethopropazine was administered, indicating that both cholinesterases must be inhibited to increase neuronal activity. In summary, these data show that in addition to AChE, intrinsic cardiac ganglia also contain distinct populations of neurons that are associated with BuChE, and the activity generated by these neurons is differentially influenced by their substrates. Because simultaneous inhibition of AChE and BuChE leads to increased neuronal activity, it is concluded that AChE- and BuChE-positive intrinsic cardiac neurons may act synergistically to influence the overall tonic activity of intrinsic cardiac ganglia.

Morphological and immunohistochemical properties of primary long-term cultures of adult guinea-pig ventricular cardiomyocytes with peripheral cardiac neurons.

Long-term (2-12 weeks) cultures of adult guinea-pig ventricular myocytes, cocultured with neurons derived from stellate or intrinsic cardiac ganglia, retain their functional properties (Horackova et al., 1993, 1994, 1995). The present study was designed to investigate the morphological and immunochemical properties of such neurons and their associated cardiomyocytes. Cultured myocytes studied by means of phalloidin-rhodamine (for F-actin) and an antibody raised against myomes revealed parallel myofibrils with striations typical of rod-shaped cardiomyocytes, even while myocytes changed from cylindrical to flattened form as they established intercellular contacts. Microtubular networks, identified by alpha-tubulin DM1A antibody, were arrayed longitudinally in myofibrils, being especially prominent during the formation of intercellular contacts between myocytes. Histochemically identified adult peripheral autonomic neurons cultured alone or with myocytes displayed a variety of shapes. alpha-Tubulin staining was associated with the somata and neurites of various-shaped neurons whether cultured alone or with myocytes. Cultured neurons derived from stellate and intrinsic cardiac ganglia also exhibited staining for the general neuronal marker PGP 9.5 (protein gene product 9.5), and for specific markers of the following neurochemicals: tyrosine hydroxylase, acetylcholinesterase, choline acetyltransferase, neuropeptide Y, vasoactive intestinal peptide, calcitonin gene-related peptide, bradykinin, oxytocin, and NADPH-diaphorase. These data indicate that: (a) adult ventricular myocytes cocultured with intrathoracic neurons retain the structural properties of adult myocytes found in vivo; (b) intrinsic cardiac and extrinsic intrathoracic neurons cultured alone or with cardiomyocytes display morphological characteristics similar to those of neurons studied in situ; (c) intrinsic cardiac and intrathoracic extracardiac neurons cultured alone or with cardiomyocytes display a variety of morphologies (unipolar, bipolar, and multipolar), larger and more multipolar neurons being present in cultures derived from stellate versus intrinsic cardiac ganglia; (d) such cultured neurons are associated with a number of neurochemicals, more than one chemical being associated with each neuron. This model presents an excellent opportunity to study the morphology of individual peripheral extracardiac and intracardiac neurons as well as their potential to produce various neurochemicals that are known to be involved in the neuromodulation of cardiomyocyte function.

Modulation of intrinsic cardiac neuronal activity by nitric oxide donors induces cardiodynamic changes.

Studies were performed to determine 1) whether a specific marker for nitric oxide production is associated with canine intrinsic cardiac neurons, 2) whether the transmembrane properties of these neurons can be altered by nitric oxide donors, 3) whether in situ intrinsic cardiac neurons are sensitive to nitric oxide donors, and 4) whether these neurons are involved in cardiac regulation. Thirty to forty percent of canine intrinsic cardiac neurons were labeled with a selective anatomic marker for nitric oxide production. Nitric oxide donors modified the transmembrane properties of a subpopulation of intrinsic cardiac neurons studied in vitro. The nitric oxide donors nitroglycerine, sodium nitrite, and nitroprusside induced concentration-dependent increases in neuronal activity frequently associated with cardiac augmentation. Similar neuronal responses were elicited by N-methyl-D-aspartate receptor activation as well as when the precursor of nitric oxide, L-arginine, and the exogenous nitric oxide donor, S-nitroso-N-acetylpenicillamine, were administered, indicating that intrinsic cardiac neurons can be modulated by nitric oxide donors. Such neurons apparently are tonically influenced by locally released nitric oxide as local administration of the competitive inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester, suppressed their spontaneous activity. These data indicate that a significant population of nitric oxide-sensitive neurons exists in the canine intrinsic cardiac nervous system that are involved in cardiac regulation.

Gross and microscopic anatomy of the canine intrinsic cardiac nervous system.

BACKGROUND: A three-dimensional description of the distribution and organization of the canine intrinsic cardiac nervous system was developed in order to characterize its full extent physiologically. METHODS: The anatomy of the canine intrinsic cardiac nervous system was investigated in 67 mongrel dogs by means of visualization following methylene blue staining as well as by light and electron microscopic analyses. RESULTS: Collections of ganglia associated with nerves, i.e., ganglionated plexuses, were identified in specific locations in epicardial fat and cardiac tissue. Distinct epicardial ganglionated plexuses were consistently observed in four atrial and three ventricular regions, with occasional neurons being located throughout atrial and ventricular tissues. One ganglionated plexus extended from the ventral to dorsal surfaces of the right atrium. Another ganglionated plexus, with three components, was identified in fat on the left atrial ventral surface. A ganglionated plexus was located on the mid-dorsal surface of the two atria, extending ventrally in the interatrial septum. A fourth atrial ganglionated plexus was located at the origin of the inferior vena cava extending to the dorsal caudal surface of the two atria. On the cranial surface of the ventricles a ganglionated plexus that surrounded the aortic root was identified. This plexus extended to the right and left main coronary arteries and origins of the ventral descending and circumflex coronary arteries. Two other ventricular ganglionated plexuses were identified adjacent to the origins of the right and left marginal coronary arteries. Intrinsic cardiac ganglia ranged in size from ones comprising one or a few neurons along the course of a nerve to ones as large as 1 x 3 mm estimated to contain a few hundred neurons. Intrinsic cardiac neuronal somata varied in size and shape, up to 36% containing multiple nucleoli. Electron microscopic examination demonstrated typical autonomic neurons and satellite cells in intrinsic cardiac ganglia. Many of their axon profiles contained large numbers of clear, round, and dense-core vesicles. Asymmetrical axodendritic synapses were common. CONCLUSIONS: The canine intrinsic cardiac nervous system contains a variety of neurons interconnected via plexuses of nerves, the distribution of which is wider than previously assumed.