Inhibition and facilitation in parasympathetic ganglia of the urinary bladder

Neurons in vesical parasympathetic ganglia receive excitatory and inhibitory inputs from both divisions of the autonomic nervous system. Sacral parasympathetic pathways (cholinergic) provide the major excitatory input to these ganglia via activation of nicotinic receptors. Parasympathetic pathways also activate muscarinic inhibitory and excitatory receptors, which may exert a modulatory influence on transmission. Cholinergic transmission is relatively inefficient when preganglionic nerves are stimulated at low frequencies (< 1 Hz). However, excitatory postsynaptic potentials (EPSPs) and postganglionic firing markedly increase during repetitive stimulation at frequencies of 1-10 Hz. It is concluded that enhanced transmitter release accounts for the temporal facilitation and that vesical ganglia function as "high pass filters" that amplify the parasympathetic excitatory input to the detrusor muscle during micturition. Transmission in vesical ganglia is also sensitive to adrenergic inhibitory and facilitatory synaptic mechanisms elicited by efferent pathways in the hypogastric nerves. The effects of exogenous norepinephrine indicate that adrenergic inhibition is mediated by alpha receptors and reflects primarily a presynaptic depression of transmitter release although postsynaptic adrenergic hyperpolarizing and depolarizing effects have also been noted. Adrenergic facilitation is mediated by beta receptors as well as unidentified receptors. Norepinephrine also can inhibit or excite spontaneously active neurons in vesical ganglia. The existence of inhibitory and facilitatory synaptic mechanisms in vesical ganglia provides the basis for a complex ganglionic modulation of the central autonomic outflow to the bladder.     

Effects of various kinds of stimulation on ornithine decarboxylase activity in superior cervical sympathetic and nodose ganglia of rats

Levels of cyclic nucleotides and ornithine decarboxylase (ODC) activity were examined following the application of various kinds of stimuli to superior cervical sympathetic ganglia (SCG), nodose ganglia, and vagus nerve fibers excised from the rat. The level of cyclic GMP in the SCG rose rapidly to about 4.5- to 7.5-fold the unstimulated control with 10 min of incubation after applications of preganglionic electrical stimulation (10 Hz), acetylcholine (ACh; 1 mM), or high extracellular K+ ( [K+]0, 70 mM). The cyclic GMP level in nodose ganglia was increased less than in the SCG by either ACh or high [K+]0 but was not affected by ACh in vagus fibers. Cyclic AMP in the SCG was also increased about 4- to 5.5-fold over the control within 10 min with the addition of ACh, norepinephrine (NE; 0.05 mM), or high [K+]0. Although NE caused a small increase in cyclic AMP, neither ACh nor high [K+]0 produced any appreciable change in nodose ganglia or vagus fibers. The ODC activity in the SCG was increased by preganglionic stimulation of 3- to 4-hr duration but not by a shorter period. A similar change in ODC activity was caused by the addition of oxotremorine (1 mM), isoproterenol (0.1 mM), NE, cyclic AMP (1 mM), or dibutyryl cyclic GMP (1 mM). The effect was exaggerated by the further addition of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. The increase in ODC activity caused by ACh was abolished by a muscarinic cholinergic antagonist, atropine (0.01 mM), and following axotomy for a week, but not by a nicotinic antagonist or by denervation in the SCG. A similar increase in ganglionic ODC activity by NE was inhibited by an adrenergic blocker, propranolol (0.01 mM), and following axtotomy for a week, but not by denervation. Cholinergic or adrenergic stimulation did not cause an increase in ODC activity in nodose ganglia or vagus fibers. These results suggest that the stimulation-induced increase in ODC activity occurs in postganglionic neurons rather than in satellite glial cells and is mediated by muscarinic cholinergic or adrenergic receptors. The process appears to involve cyclic nucleotide-mediated protein biosynthesis in the SCG.     

In vitro effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland

1. To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10-1,000 nM NPY in the presence or absence of 10 microM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA). 2. NPY (10-1,000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1,000 nM), enhanced NE stimulation of melatonin production. 3. NPY (1,000 nM) impaired 3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with 3H-NE. 4. These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.     

Pineal muscarinic phosphoinositide response: pertussis toxin resistant signaling with very low receptor number

Autoradiography revealed very low density of muscarinic receptors in the rat pineal gland. Yet, the magnitude of phosphoinositide hydrolysis elicited with 0.1 mM carbachol was similar to that seen with 1 mM norepinephrine. The cholinergic and adrenergic phosphoinositide responses were fully additive. The cholinergic signal was insensitive to pertussis toxin both in vivo and in vitro and persisted in pineals cultured for 5 days. The data expand our previous finding on functional muscarinic acetylcholine receptors in the rat pineal gland.     

Developmental study of ouabain inhibition of adrenergic induction of rat pineal serotonin N-acetyltransferase (EC 2.3.1.87)

The activity of arylalkylamine N-acetyltransferase (EC 2.3.1.87), the rate-controlling enzyme in melatonin synthesis is stimulated approximately equal to 100-fold by an adrenergic cyclic AMP mechanism in both neonatal and adult rat pineal glands. This stimulation is blocked in the adult gland by the depolarizing agents ouabain (1 microM) and K+ (80 mM) (Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K., and Marks, B.H. (1975) Mol. Pharmacol. 11, 241-255). In the present study pineal glands obtained from prenatal to adult rats were used; it was found that K+ (80 microM) inhibited the adrenergic stimulation of N-acetyltransferase activity at all ages but that ouabain (1 nM to 1 mM) treatment was not inhibitory early in development. In contrast, in the neonate, ouabain (1-100 nM) enhanced adrenergic induction of N-acetyltransferase activity, and ouabain treatment alone (1-1000 nM) stimulated N-acetyltransferase activity. A small stimulation was also seen at one concentration (1 nM) in the adult. Analysis of the development of high affinity ouabain binding sites and Na+,K+-ATPase activity in the intact pineal gland indicated that the developmental pattern of both resemble the development of ouabain inhibition of the adrenergic stimulation of N-acetyltransferase activity. All are low for the first few days of life, gradually increase during the next 3 weeks of life, and then approach adult levels. Similarly, ouabain (1 nM to 1 mM) had no effect on 86Rb uptake in the 2-day-old gland but blocked (IC50 congruent to 20 nM) 86Rb uptake in the adult gland. These findings indicate ouabain probably has little inhibitory effect on the norepinephrine stimulation of N-acetyltransferase activity in the neonatal because a high affinity ouabain binding form of Na+,K+-ATPase activity, similar to the alpha + form identified in rat brain, is at very low levels in the pinealocyte. Accordingly, it appears that an ouabain-insensitive mechanism in the neonatal gland maintains membrane potential and that this mechanism plays a less important role in the adult. The explanation of why ouabain alone stimulates N-acetyltransferase activity and why it enhances the effects of norepinephrine in the neonatal pineal gland might be that ouabain acts on surviving neural elements present in the gland to cause the net release of a transmitter, perhaps norepinephrine, which then stimulates N-acetyltransferase activity.     

A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons.

Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.     

Effects of age on the adrenergic cardiac neuroeffector junction

Although it is clear that adrenergic nervous system control of cardiac function decreases with age and that the effector organ fails to adjust to this decreased control, it is not completely evident which of the many mechanisms operant at the adrenergic-cardiac neuroeffector junction contribute to this state. Prejunctionally, it appears that norepinephrine content decreases with age and that adrenergic axonal degeneration occurs. Also, evidence is available to suggest that modulation by prejunctional alpha adrenergic receptors of norepinephrine release is altered with increasing age, as is neuronal uptake of norepinephrine. Postjunctionally, it appears that beta-adrenergic receptor sensitivity to agonists undergoes age-related alterations, and possibly post receptor mechanisms involved in receptor-response coupling. Other mechanisms, such as those involved in transmitter uptake into extraneuronal sites, adrenergic neuronal responsiveness to stimulation, transmitter release and turnover, calcium and prejunctional receptor modulation of transmitter release, postjunctional receptor development of supersensitivity or subsensitivity, need further elucidation in order to have an understanding of the factors that contribute to the breakdown of homeostatic mechanisms that regulate the heart.     

Regulation of chicken pineal arylalkylamine-N-acetyl transferase by postsynaptic alpha 2-adrenergic receptors

The present investigation sought to characterize the adrenergic inhibition of arylalkylamine-N-acetyltransferase in cultured chicken pineal glands. Arylalkylamine-N-acetyltransferase, the melatonin rhythm generating enzyme, displays daily oscillations of activity that are driven by a circadian oscillator. Norepinephrine released at sympathetic nerve endings inhibits the enzyme and appears to play a role in maintaining a circadian rhythm of melatonin release. Chicken pineal glands were isolated in organ culture and the effects of adrenergic agents on the night time peak of N-acetyltransferase activity were studied. Norepinephrine and clonidine prevented 50 to 65% of the nocturnal rise of N-acetyltransferase activity. When applied at middark, norepinephrine and clonidine caused a 50 to 65% inhibition of N-acetyltransferase activity in 2 hours. Dose-response studies indicated clonidine was 100 times more potent than norepinephrine or cirazoline at inhibiting N-acetyltransferase activity. Inhibition of N-acetyltransferase activity was also observed, at micromolar concentration with epinephrine, UK 14,304 and alpha-methylnorepinephrine but not with phenylephrine, isoproterenol or dopamine. Epinephrine and clonidine actions were antagonized by yohimbine but not by prazosin. Destruction of the presynaptic compartment by bilateral superior cervical ganglionectomy did not affect the clonidine-induced inhibition of N-acetyltransferase and its reversal by yohimbine. It is concluded that the adrenergic inhibition of N-acetyltransferase activity in chicken pineal gland probably occurs via stimulation of postsynaptic alpha 2-adrenergic receptors.     

Presynaptic alpha-receptor control of adrenergic transmitter release in blood vessels

The presynaptic alpha-adrenergic receptor control of transmitter release in vascular tissues is discussed. A model of adrenergic innervation of the vascular bed is proposed based on ultrastructural and histochemical evidence. Evidence is presented to support the concept of intermittent or periodic release of norepinephrine (NE) from the varicosity. Intermittency combined with a mechanism such as presynaptic control to ensure spatial distribution of release sites, along with a slow effector response and recovery, results in a smooth, generalized change in tone and an overall economy of transmitter. The effective concentration of NE around the presynaptic membrane is maintained for considerably less than 0.1 s. It is argued that the transient presence of transmitter in the synapse combined with intermittency of release does not favor accumulation of transmitter at the cleft at physiological frequencies or desensitization of presynaptic receptors. In addition, intermittency provides an explanation for why exogenous NE is more effective presynaptically in influencing release than endogenous NE. The importance of cleft width in presynaptic control of transmitter release, the possible complications caused by facilitation, and resolution of some apparent problems with the presynaptic hypothesis are also discussed.     

Lanthanum as a tool to study the role of phosphatidylinositol in the calcium transport in rat parotid glands upon cholinergic stimulation.

We describe the effects of lanthanum on protein secretion, potassium efflux, calcium uptake and phosphatidylinositol turnover stimulated by cholinergic agonists in rat parotid glands. Carbachol increases in vitro calcium uptake, protein secretion and K+ efflux through muscarinic receptor; however it fails to stimulate protein discharge or K+ release in a incubation medium free of calcium. Lanthanum inhibits calcium uptake, protein secretion and K+ efflux induced by carbachol without impairing protein discharge stimulated by norepinephrine through the beta-adrenergic receptor. Norepinephrine, in the presence of calcium in the incubation medium, stimulates the K+ efflux through the alpha-adrenergic receptor: this effect is suppressed by lanthanum. These results emphasize the role of increased influx of calcium in the cellular phenomena controlled by muscarinic or alpha-adrenergic receptors. Carbachol increases phosphatidylinositol turnover in the absence of calcium in extracellular medium; indeed it is shown that carbachol increases the rate of phosphatidylinositol breakdown and that lanthanum impairs this cholinergic effects. From these data it is suggested that the interaction between cholinergic agonist and muscarinic receptor could induce a stimulation of 'phosphatidylinositol turnover' which could control the calcium influx according to the gradient through the plasmalemma membrane.     

Nonclassical synaptic functions of transmitters

Nonclassical synaptic functions are considered in two groups, mainly by reference to the models provided by sympathetic ganglia. Slow postsynaptic potentials (PSPs) are compared with classical fast PSPs. Features include loose delivery of transmitter to receptor, very long synaptic delays and durations of PSPs, slow removal of transmitter or of its effects, integration of repetitive inputs, electrogenesis without large increases in ionic conductances. Neuromodulatory actions affect synaptic efficacy without direct excitatory or inhibitory responses to the transmitter. These include a) control of presynaptic release, and b) contingent postsynaptic actions. In b, a modulatory transmitter alters the efficacy of action by another transmitter. The alteration may persist long after exposure to the modulatory transmitter; in mammalian sympathetic ganglia, exposure to dopamine or to a conditioning train of preganglionic volleys induces a long-term enhancement of the muscarinic slow excitatory PSP. Or the alteration may be restricted mostly to the presence of a modulatory transmitter, with examples cited. Nonclassical synaptic functions may be providing revolutionary possibilities for dealing with slow and broadly distributed cerebral functions, manifested electrophysiologically and behaviorally, that have been difficult to analyze successfully in terms restricted to the fast and discretely localized classical synaptic functions.     

Support for a role for feedback regulation of norepinephrine release

There is abundant evidence that norepinephrine (NE) and other sympathomimetic amines with alpha-adrenoceptor activity inhibit the electrically evoked release of NE, whereas phenoxybenzamine and other alpha-adrenergic blocking agents enhance the electrically evoked release of NE. The physiological relevance of these observations, however, is disputed. The intent of this paper is to show that alpha-adrenergic blocking agents generally enhance transmitter output on nerve stimulation, but that some are more selective for presynaptic than for postsynaptic alpha receptors. Suggestions are made to account for the modulation of NE release as evoked by a single pulse.     

Beta- and alpha-adrenergic receptors are involved in regulating type II thyroxine 5'-deiodinase activity in the rat Harderian gland.

The role of alpha- and beta-adrenergic receptors in regulation of rat Harderian gland type II thyroxine 5'-deiodinase (5'-D) activity was investigated. Our results show that isoproterenol, a beta-adrenergic agonist, and phenylephrine, an alpha-adrenergic agonist, elicited increases in Harderian gland 5'-D activity. The activation was dependent on the time and the dose of the drug. Other adrenergic agonists, i.e., norepinephrine, methoxamine or terbutaline, also clearly increased the enzyme activity. Moreover, administration of propranolol, a beta-adrenergic blocker, or prazosin, an alpha-adrenergic blocker, completely prevented the activation of the enzyme induced by norepinephrine. Results show a clear regulation by adrenergic mechanisms of 5'-D activity in the rat Harderian gland, where alpha- and beta-adrenergic receptors appear to be involved.     

Postsynaptic localization of the alpha receptor-mediated stimulation of phosphatidylinositol turnover in pineal gland.

The phosphatidylinositol effect in rat pineal gland (defined as the enhanced incorporation of P_i into phosphatidylinositol which is elicited by agonists) is mediated through alpha-adrenergic receptors. Experiments with glands from animals treated with 6-hydroxydopamine, with dispersed pinealocytes and with a number of relatively specific pre- and postsynaptic alpha-adrenergic agonists and antagonists have shown the receptors involved to be located at postsynaptic sites.     

Alpha-adrenergic receptor modulation of the intrathoracic efferent sympathetic nervous system regulating the canine heart

The augmentation of ventricular inotropism induced by electrical stimulation of acutely decentralized efferent sympathetic preganglionic axons was reduced, but still present, following administration of hexamethonium (10 mg/kg i.v.). While hexamethonium continued to be administered, the cardiac augmentations so induced were enhanced significantly following administration of the alpha-adrenergic receptor blocking agent, phentolamine myselate (1 mg/kg i.v.). Stimulation of the sympathetic efferent postganglionic axons in cardiopulmonary nerves induced cardiac augmentations that were unchanged following administration of these agents singly or together. The cardiac augmentations induced by stimulation of efferent preganglionic sympathetic axons were unchanged when phentolamine was administered alone. The augmentations of cardiac inotropism induced by efferent postganglionic sympathetic axonal stimulation were decreased following local administration of the beta-adrenergic antagonist timolol into the ipsilateral stellate and middle cervical ganglia. Thereafter, these augmentations were unchanged following the subsequent intravenous administration of phentolamine. It is concluded that the activation of cardiac neurons in the stellate and middle cervical ganglia by stimulation of efferent preganglionic sympathetic axons can be modified by alpha-adrenergic receptors and that these effects are dependent upon beta-adrenergic receptors, not nicotinic ones, in intrathoracic ganglia.     

Quantal Mechanism of Transmitter Release during Progressive Depletion of the Presynaptic Stores at a Ganglionic Synapse : The action of hemicholinium-3 and thiamine deprivation

In the present experiments we interfered with the mechanism of acetylcholine (ACh) synthesis in the rat superior cervical ganglion by impairing the supply of either the choline group (hemicholinium no. 3 [HC-3]treatment) or the acetyl group (thiamine deprivation). Under both conditions stimulation causes in the ganglion a progressive decline in ACh output associated with a depletion of transmitter tissue content. ACh release from the terminals of a single preganglionic fiber was estimated from the quantum content value of the evoked excitatory postsynaptic potentials (EPSP's) recorded intracellularly in the ganglion neuron under test. The present observations indicate that Poisson statistics describe transmitter release at either low or high release levels. Furthermore, the progressive decline in the rate of ACh output occurring during repetitive stimulation is shown to correspond to a progressive decrease in the number of transmitter quanta released per impulse and not to any modification in the size of individual quanta. Some 8,000 transmitter quanta proved to represent the presynaptic transmitter store initially present in those terminals on a neuron that are activated by stimulation of a single preganglionic fiber. Speculations are considered about synaptic efficacy and nerve connections in rat autonomic ganglia. It is suggested that six preganglionic fibers represent the mean input to a ganglion neuron.     

ACETYLTRIETHYLCHOLINE: A CHOLINERGIC FALSE TRANSMITTER IN CAT SUPERIOR CERVICAL GANGLION AND RAT CEREBRAL CORTEX

The accumulation and metabolism of [¹⁴C]triethylcholine by cat superior cervical ganglia [rested or stimulated (20 Hz)] and by rat cerebral cortex minces was measured. In ganglia, preganglionic nerve stimulation increased the accumulation (2.4 fold) and the acetylation (5.7 fold) of triethylcholine; however the depletion of the ganglion's acetylcholine content was 9.5 times greater than the amount of acetyltriethylcholine synthesized. In the presence of eserine, neither stimulated nor rested ganglia synthesized any extra (surplus) acetyltriethylcholine. It is concluded that the rate-limiting step in acetyltriethylcholine synthesis is the acetylation of triethylcholine by choline acetyltransferase. Subsequent preganglionic nerve stimulation of ganglia, which had been stimulated during the exposure to [¹⁴C]-triethylcholine, caused the increased release of only acetyltriethylcholine; the release was frequency-dependent, required the presence of Ca²⁺, and was blocked by increasing the ratio of Mg²⁺/Ca²⁺ in the perfusion fluid. All of the acetyltriethylcholine which had been accumulated was available for release. Rat cerebral cortex also accumulated triethylcholine and acetylated about 3% of the accumulated choline analogue. Subsequent stimulation by high K⁺ (46 mM)-atropine (3 μM) caused the increased release of acetyltriethylcholine from the cortex and this release required the presence of Ca²⁺. Triethylcholine can therefore form a cholinergic false transmitter in the cat superior cervical ganglion and the rat cerebral cortex.     

Evidence of alpha 1-adrenergic----protein kinase C----Na+/H+ antiporter-dependent increase in pinealocyte intracellular pH. Role in the adrenergic stimulation of cGMP accumulation

The regulation of intracellular pH (pHi) in isolated rat pinealocytes was studied using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.09 when the extracellular pH (pHe) was 7.2. Treatment of pinealocytes with the physiological regulator of pineal function, norepinephrine, resulted in a concentration-dependent increase in pHi. Further analysis indicated that norepinephrine is probably acting via an alpha 1-adrenergic----[Ca2+]i----Ca2+/phospholipid- dependent protein kinase (protein kinase C) mechanism to activate the Na+/H+ antiporter, thereby causing cytoplasmic alkalization. A potential influence of cytosolic alkalization on the responsiveness of cyclic nucleotides to adrenergic agonists was also studied. Five analogs of the antiporter inhibitor amiloride reduced norepinephrine stimulation of cGMP accumulation with the same relative potency as they act on the antiporter. In contrast, although inhibitory effects of these compounds on cAMP accumulation were detectable, they occurred at 10-100-fold higher concentrations, and the relative potency of these inhibitors did not indicate they were acting via the antiporter. These findings provide evidence that 1) alpha 1-adrenergic receptor activation increases pinealocyte pHi through Ca2+----protein kinase C-dependent activation of the Na+/H+ antiporter; and 2) norepinephrine stimulation of cGMP accumulation is pHi-dependent. It would appear that alpha 1-adrenergic regulation of pHi via the Na+/H+ antiporter may be of general importance in the control of cGMP accumulation.     

海马内微量注射褪黑素的免疫调节及机制研究

本文采用海马内微量注射褪黑素（ｉｈ,ＭＴ）的技术,发现ｉｈＭＴ具有明显的免疫调节作用。进一步发现,ｉｈ ＭＴ能提高海马内去甲肾上腺素水平,通过α２受体,交感神经系统调节免疫功能。本研究还发现,脾功能能产生ＭＴ,脾细胞中可能存在着ＭＴ合成的关键酶。ｉｈＭＴ能通过交感神经系统去甲肾上腺素能神经,促进脾细胞产生ＭＴ,由此诱导β－Ｅｎｄ调节免疫功能,ｉｈＭＴ还能通过海马去甲肾上腺素作用α２受体,反馈抑制松     

Cholinergic signaling in the rat pineal gland

Summary 1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotoninN-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion.2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland.3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs).4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.