Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo.

Previous reports indicate that alterations of activity of cholinergic neurons $\text{in vivo}$ are followed by parallel changes in sodium-dependent high affinity choline uptake $\text{in vitro}$. These results are consistent with the proposal that this portion of choline uptake is regulatory in the synthesis of ACh. These results also suggest the possibility of utilizing sodium-dependent high affinity choline uptake as a measure of the relative state of cholinergic activity $\text{in vivo}$. In this study, we administer a number of drugs reported to alter turnover and release of ACh (both are measures of cholinergic activity $\text{in vivo}$, and subsequently examine sodium-dependent high affinity choline uptake $\text{in vitro}$. Administration of pentobarbital, chloral hydrate, morphine, physostigmine, Δ⁹ THC, hemicholinium-3 and oxotremorine, drugs which decrease ACh turnover and release, caused a reduction in choline uptake. Conversely, administration of pentylenetetrazol, atropine, scopolamine, and haloperidol, drugs which increase ACh turnover and release, caused an increase in choline uptake $\text{in vitro}$. These findings support the proposal that sodium-dependent high affinity choline uptake can be used as a relative measure of the activity of cholinergic neurons $\text{in vivo}$.     

Kinetics of in vivo binding of antagonist to muscarinic cholinergic receptor in the human heart studied by positron emission tomography

Positron Emission Tomography (PET) was used to analyse $\text{in vivo}$ antagonist binding to human myocardial muscarinic cholinergic receptor. The methiodide salt of the muscarinic antagonist, quinuclidinyl benzilate (MQNB), was labeled with the positron emitter, Carbon-11, and injected intravenously to 8 normal subjects. ¹¹C-MONB concentration was determined $\text{in vivo}$ in the ventricular septum from 40 cross-sectional images acquired at the same transverse level over a period of 70 minutes. In 4 subjects, various amounts of unlabeled atropine were rapidly injected at 20 minutes to study whether atropine competitively inhibited MQNB.The kinetics of binding of ¹¹C-MQNB were not the same $\text{in vivo}$ and $\text{in vitro}$. The apparent dissociation rate of ¹¹C-MQNB $\text{in vivo}$ was much slower (by 1 to 2 orders of magnitude) than that observed $\text{in vitro}$ with ³H-QNB. After atropine injection, ¹¹C-MNQB dissociated from its binding sites at a rate that apparently depended on the amount of atropine present. ¹¹C-MQNB kinetics were analysed with a mathematical model which assumes the existence of a boundary layer containing free ligand in the vicinity of the binding sites. The dissociation rate of the radioligand depends on the probability of its rebinding to a free receptor site.     

Further studies on the pharmacology of a false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine).

The pharmacology of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine, DEC) was studied with various preparations. It was found to inhibit the neuromuscular transmission of frog sciatic nerve-gastrocnemius muscle $\text{in}$$\text{vitro}$ with ED₅₀ of 1.93 (0.66 - 5.79) × 10⁻⁴ M. DEC was also found to inhibit dog chorda tympani-Wharton's duct (postganglionic parasympathetic neuro-effector junction) and cat superior cervical ganglionnictitating membrane (sympathetic ganglion) preparations $\text{in}$$\text{vivo}$ with ED₅₀'s of 6.2 (1.8 – 21.1) mg/kg and 12.0 (5.7 - 25.2) mg/kg, respectively. After blockade of these preparations with DEC, the former was still responsive to intravenous injection of pilocarpine (1 mg/kg) and choline (10 mg/kg) and the latter to close arterial injection of acetylcholine (100 μg/injection) and choline (3 mg/min infusion). These results support the idea that DEC paralyzes cholinergic neurons possibly through false cholinergic transmission without blocking the cholinergic receptor at the post-junctional membrane.     

On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary

An $\text{in}$$\text{vitro}$ perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release ($\text{IC}{}_{50}{}^1\text{= 6 × 10}{}^{\mathrm{?8}}\text{M and}\text{10}{}^{\mathrm{?5}}\text{M}$ respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.     

Rat posterior lobe modulates growth hormone release in vivo and in vitro

Electrical stimulation of hypophysical posterior lobes $\text{in vivo}$ evokes a significant decrease of plasma growth hormones (GH) and an increase of plasma corticotropin (ACTH) concentrations. Electrical stimulation of posterior lobes $\text{in vitro}$ evokes the simultaneous release of GH inhibiting factor(s) (GHRIF) and ACTH releasing factor(s) (CRF) into the medium. Pretreatment of media with thioglycolate abolishes the CRF and GHRIF activity, but reveals GH releasing factor(s) (GHRF). Median eminence extracts and vasopressin have potent GHRF and CRF activity. Vasopressin may account fully for the CRF and partially for GHRF activity. Results suggest that hypothalamo-neurohypophysical axons release GHRIF, vasopressin and possibly a GHRF into a portal circulation to modulate the secretion of GH and ACTH.     

Fast cation flux from Torpedocalifornica membrane preparations: Implications for a functional role for acetylcholine receptor dimers

Closed membrane vesicles derived from the innervated face of $\text{Torpedo}$$\text{californica}$ electroplax respond to the cholinergic agonist carbamylcholine by a rapid efflux of cations. This response is detected by release of ²²Na enclosed within the vesicles and is considerably faster than previously reported for this $\text{in}\text{vitro}$ system. It is considered likely that the rapid response is analogous to physiological phenomena since it has the appropriate pharmacological characteristics; it desensitizes upon prolonged contact with the agonist and it has a dose-response curve in a physiological range. It is further shown that a dimeric form of the acetylcholine receptor, stabilized by chemical modification methods, is fully active in terms of the carbamylcholine elicited response.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

Cholinoceptor-mediated control of catecholamine release from chromaffin cells in the American eel,Anguilla rostrata

The cholinergic agonist-induced secretion of catecholamines from chromaffin cells in the American eel, Anguilla rostrata, was assessed using a salineperfused posterior cardinal vein preparation. Direct membrane depolarization with 60 mmol·l^-1 K⁺ caused a significant release of catecholamines (adrenaline + noradrenaline) into the perfusate which was unaffected by pre-treatment with the ganglion blocker, hexamethonium (final concentration = 10^-3 mol · l^-1). The nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, evoked catecholamine release in response to several doses exceeding 10^-7 mol; at 10^-5 mol the response was abolished by pre-treatment with the ganglion blocker, hexamethonium (final concentration = 10^-3 mol · l^-1). The muscarinic receptor agonist, pilocarpine, did not elicit catecholamine release in response to any of the doses administered (10^-8–10^-4 mol). A single injection of the mixed nicotinic/muscarinic cholinoceptor agonist, carbachol (10^-5 mol), caused the release of catecholamines which was abolished by pre-treatment with hexamethonium but which was unaffected by pre-treatment with the muscarinic receptor antagonist atropine (final concentration = 10^-5 mol · l^-1). The results of this study indicate that the process of cholinergic agonist-induced catecholamine secretion from the chromaffin cells in the American eel is mediated exclusively by activation of nicotinic receptors with no involvement of the muscarinic receptor.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium iodide - MS222 ethylaminobenzoate - HPLC high-performance liquid chromatography - PCV posterior cardinal vein - SEM standard error of the mean     

Lack of growth hormone-releasing activity of (pyro)Glu-Ser-Gly-NH2

Youdaev $\text{et al.}$ (1) reported that (pyro)Glu-Ser-Gly-NH₂ isolated from bovine hypothalami or made synthetically stimulates the release of growth hormone (GH). Therefore, we synthesized the tripeptide and tested it in several $\text{in vivo}$ and $\text{in vitro}$ assay systems for GH releasing activity. Our results demonstrate that (pyro)Glu-Ser-Gly-NH₂ does not stimulate the release of immunoreactive GH from rat pituitaries $\text{in vitro}$ in doses of 0.1–1000 nanog/ml. Similarly, (pyro)Glu-Ser-Gly-NH₂, injected intravenously into rats in doses of 1μg and 10μg/rat or infused into a hypophysial portal vessel in doses of 0.01 μg and 0.1 μg/rat did not increase serum GH levels as measured by radioimmunoassay. When this tripeptide was injected intravenously in doses of 500 μg into sheep it did not raise plasma GH levels. These results demonstrate that under the conditions of this investigation (pyro)Glu-Ser-Gly-NH₂ does not display any GH-releasing activity.     

Identification and purification of factor B-GHRH from hypothalami which releases growth hormone

Exploratory purifications and assays of fractions for release of growth hormone (GH) revealed two separable entities each of which unambiguously released GH by radioimmunoassay. They are provisionally designated factor A-GHRH and factor B-GHRH until they are chemically and biologically characterized. After initial steps of isolation from porcine hypothalami, factor B-GHRH was extensively purified by stepwise chromatography using Bio-Gel P-2, Sephadex LH-20, Sephadex G-25 with a partition system of acetic acid-butanol-pyridine, DEAE-Sephadex A-25, and Bio-Gel CM-2. Assays showed that certain fractions were active, $\text{in vitro}$, at levels of $\text{ca}$. 15 μg. Factor B-GHRH is inhibited by somatostatin.     

Potassium release from submandibular salivary gland in vitro

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K⁺ in response to α-adrenergic and muscarinic cholinergic stimulation. The system employed the specific α-, β-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists l-epinephrine and carbamylcholine both of which required the presence of Ca²⁺ for their effect. The introduction of Ca²⁺ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K⁺ release. Ouabain also allowed extensive K⁺ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K⁺ movement. K⁺ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca²⁺. The implication of cyclic GMP at some stage of K⁺ release was suggested by experiments with a phosphodiesterase inhibitor.The results support an hypothesis where the initial stimulus at either α-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca²⁺ enters the cells resulting in K⁺ release. The loss of K⁺ is quickly countered by the ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$) ATPase which would be activated by the lowered intracellular K⁺ levels.     

Effects of naloxone on the secretion of prolactin and corticosterone induced by 5-hydroxytryptophan and a serotonergic agonist, mCPP

The effects of naloxone, an opiate “pure” receptor antagonist, on the release of prolactin and corticosterone in the rat were studied following the administration of the serotonin precursor 5-hydroxytryptophan or the serotonin receptor agonist (?) -$\text{m}$-chloropnehylpiperazine. Naloxone clearly antagonizes the release of prolactin induced by 5-hydroxytryptophan administered alone at a dosage of 50 mg/Kg/b.wt. or at dosage of 30 mg/Kg/b.wt. preceded 60 minutes before injection by the administration of the serotonin uptake blocker fluoxetine. The opiate antagonist does not modify the increase in blood level of prolactin induced by (?) ?$\text{m}$-chlorohenylpiperazine. Naloxone itself does not reduce the increase in plasma level of corticosterone induced by 5-hydroxytryptophan, 5-hydroxytryptophan +fluoxetine or (?)?$\text{m}$-chlorophenylpiperazine.The results suggest that endogenous opioids may be involved in the increase in serum level of prolactin induced by 5-hydroxytryptophan and also indicate the existence of different serotonergic neurotransmitter circuits capable of modulating the release of prolactin and corticosterone. A mutual interplay between serotonergic and opiate neurons may be involved in controlling the release of prolactin, but such an interplay does not seem to occur in the secretion of corticotrophin-releasing hormone.     

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular mucin of the mouse

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [³H]leucine and $\text{N-}\text{acetyl}\text{[}{}^{14}\text{C}\text{]}\text{mannosamine}$ into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [³H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [¹⁴C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [¹⁴C]ManNAc relative to [³H]leucine into submandibular mucin of the mouse. During a period of 10 h the [¹⁴C]ManNAc incorporation into the mucin was enhanced 2–3-fold by isoproterenol and 3–4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.     

Isolation by affinity chromatography of a cholinergic proteolipid from Nucleus caudatus.

A cholinergic proteolipid fraction (i.e. a hydrophobic lipoprotein) was separated from the $\text{n. caudatus}$ of the cow, using affinity chromatography with the lipophilic gel Sephadex LH-20 and p-phenyltrimethylamonium as the active group. High affinity binding studies showed that only the specific fraction, desorbed after an acetylcholine (or acid) pulse, and corresponding to 0,72% of the proteolipids, is the one that binds the cholinergic ligands. The binding of (³H)atropine and (¹⁴C)d-tubocurarine demonstrated that there are 814 picomoles/g fresh tissue of muscarinic sites and only 76 picomoles/g of nicotinic sites. The specific radioactivity for (³H)atropine is 10,000 nmoles/g protein, suggesting a high degree of purification of the specific cholinergic proteolipid.     

Interactions of lead and calcium on the synaptosomal uptake of dopamine and choline

Exposure of rodents to lead $\text{in vivo}$ has been associated with alterations in cholinergic and dopaminergic neurotransmission in the CNS. These effects have been hypothesized to result from competitive interactions between lead and calcium at sites involved in uptake and release of neurotransmitters and their precursors. These experiments reproduced the $\text{in vivo}$ observation by $\text{in vitro}$ exposure of crude synaptosomal suspensions to lead. Lead-induced inhibition of high affinity choline uptake was mimicked by reduced in vitro calcium concentrations, which suggests that lead's effects on cholinergic function are explainable by the lead-calcium hypothesis. However, inhibition of dopamine uptake was produced only by lead and not by reduced calcium; further additions of calcium did not reverse lead-induced effects on dopamine uptake. Increased calcium concentrations were shown to increase the release of dopamine; lead in the presence of normal calcium concentration did not affect dopamine release. However, more dopamine was released when increased calcium was combined with exposure to 1 × 10^?4 lead. This effect may have resulted from lead's ability to increase the uptake of calcium by synaptosomes. Thus, the interactions between lead and calcium appear to differ in terms of effects on cholinergic and dopaminergic function; in the former, the results suggest a competitive interaction similar to that shown functionally at peripheral cholinergic sites; in the latter, a different role for calcium is hypothesized which may account for the different effects of lead.     

Effects of neurotransmitters and other pharmacological agents on 32Pi incorporation into phospholipids of the iris muscle of the rabbit

The effects of norepinephrine, other catecholamines, α- and β- adrenergic receptor blocking agents and acetylcholine on the incorporation of ³²Pi into phospholipids of the iris muscle of the rabbit were studied $\text{in vitro}$. There was a marked stimulation of ³²Pi into phosphatidic acid (PhA), phosphatidyl inositol (PhI) and to a much lesser extent phosphatidyl choline but not into phosphatidyl ethanolamine. The increase in the ³²P labeling of PhA and PhI in the presence of norepinephrine or acetylcholine, which ranged from 2- to 6-fold, was found to be time- and concerntration-dependent. Under our experimental conditions, several adrenergic drugs, including DL-propranolol, phentolamine, isoproterenol, phenylephrine, but not sotalol, increased markedly (nearly up to 5-fold) the ³²Pi incorporation into PhA and PhI of the iris. In contrast, phenoxybenzamine, an α-receptor blocker, blocked completely the stimulatory effects of norepinephrine on phospholipid synthesis. The stimulation of phospholipid synthesis by acetylcholine was completely abolished by atropine. Incorporation of ³²Pi into PhA and PhI was significantly increased in the presence of serotonin, dopamine, epinephrine or histamine. Addition of γ-aminobutyric acid or cyclic AMP was ineffective. These observations suggest that in the iris muscle of the rabbit, which is innervated by cholinergic and adrenergic fibers, the phospholipid effect is probably a membrane effect that is not associated with synaptic transmission.     

The identification of an invertebrate acetylcholine receptor

The results of a series of experimental studies have culminated in the identification of an acetylcholine receptor from the invertebrate $\text{Limulus polyphemus}$. The binding ligand α-bungarotoxin was used to identify a specific protein in the central nervous system tissue of this organism. The specific interaction of α-bungarotoxin with an acetylcholine receptor has been confirmed by physiological, competitive binding, subcellular fractionation and autoradiographic techniques. The toxin binding protein was solubilized and exhibited properties consistent with the nature of a nicotinic cholinergic receptor. Therefore, the identified protein is proposed as an acetylcholine receptor protein from the central nervous system of this invertebrate species.     

Release of endogenous dopamine from tuberoinfundibular neurons

Release of endogenous dopamine (DA) from arcuate-periventricular nucleus-median eminence fragments has been analyzed in an $\text{in vitro}$ static incubation system.Exposure of these hypothalamic fragments to increasing concentrations of K⁺ ions produced a dose-dependent release of endogenous DA. The highest rate of K⁺-stimulated DA efflux occurred in the first 10 minutes, thereafter it progressively decline reaching prestimulated levels at 30 minutes. If two consecutive depolarizing stimuli of 40 mM KCl were applied to the same hypothalamic fragment, after a 40 minutes rest period, an equivalent release of endogenous DA occurred. Removal of Ca⁺⁺ ions from the incubation medium containing the Ca⁺⁺ chelator EGTA caused a decrease of basal DA efflux and completely prevented the K⁺-induced release of DA.Furthermore when verapamil, a blocker of Ca⁺⁺ entrance, was added to the incubation medium in a concentration of 50 μM, the K⁺-induced DA efflux was completely counteracted, whereas spontaneous release was unmodified.Finally nomifensine, a potent blocker of DA uptake, added $\text{in vitro}$ in a final concentration of 10 μM, significantly reinforced K⁺-induced release of endogenous DA. Since nomifensine did not modify basal DA release, this study confirmed its prevalent uptake blocking property rather than its releasing action on DA.     

Antinociceptive and hypothermic effects of trimethyltin

Trimethyltin (TMT) induced a dose-dependent antinociceptive and hypothermic effect in mice. Antinociception was not attenuated by naloxone but was reversed by atropine. TMT, however, was ineffective in displacing (³H)-QNB binding $\text{in vitro}$ and did not affect (³H)-QNB binding or acetylcholinesterase activity after $\text{in vivo}$ administration. The ethyl ester of nipecotic acid, a specific inhibitor of synaptosomal GABA uptake, exerted a similar antinociceptive effect that could be blocked by atropine. The GABA receptor antagonist bicuculline attenuated antinociception induced by TMT and nipecotic acid ethyl ester but not by morphine or oxotremorine. γ-Vinyl GABA, an irreversible inhibitor of GABA metabolism, prolonged TMT but not morphine-induced antinociception. In contrast, neither the dose-response nor the time course of TMT-induced hypothermia were affected by any of the drugs tested. The findings suggest that the GABAergic system may be involved in TMT induced antinociception; however, the mechanism responsible for the hypothermic effect of TMT is not apparent.