Studies on action mechanisms of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium

(2-Hydroxyethyl) methyldiethylammonium (DEC; Diethylcholine) was found to inhibit cholinergic fibers slowly, both in skeletal muscle (ED₅₀: 2.25 × 10^?5 M in chick biventer cervicis and 42 mg/kg in rat sciatic-gastrocnemius) and in smooth muscle preparations (ED₅₀: 7.7 × 10^?4 M in transmurally stimulated guinea-pig ileum) without having any effect on dose-response curves of acetylcholine to contract chick biventer cervicis, frog rectus abdominis and guinea-pig ileum. These results indicate that DEC acts at the prejunctional nerve fibers, but not at the postjunctional cholinergic receptor sites. DEC was acetylated efficiently both by choline acetyltransferase and by minced rat brain, suggesting that it can be acetylated to acetyl-DEC in the nerve ending. Acetyl-DEC was found to block acetylcholine actions competitively both in smooth and in skeletal muscle preparations (1 × 10^?3 ? 1 × 10^?2M) indicating that the acetylated product of DEC can serve as an antagonist at the cholinergic receptor site. It is therefore concluded that DEC is a false cholinergic transmitter.     

Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of cortisol and cortisone.

Superior antitumor activity of 1-β-D-arabinofuranosylcytosine (ara-C) conjugates of prednisolone and prednisone against L1210 leukemic mice, based on ara-C content, has encouraged us to synthesize 5′-(cortisone-21-phosphoryl)-1-β-D-arabinofuranosylcytosine (I) and 5′-(cortisone-21-phosphoryl)-1-β-d-arabinofuranosylcytosine (II) by condensation of N⁴,2′,3′-triacetyl-1-β-d-arabinofuranosylcytosine 5′-monophosphate with cortisol and cortisone in the presence of N,N′-dicyclohexylcarbodiimide at room temperature followed by removing the acetyl groups in 2 N methanolic ammonia in 20% yield. The conjugates I and II inhibited the $\text{in}\text{vitro}$ growth of L1210 by 50% (ED₅₀) at 0.25 μM and 0.07 μM, respectively, while ara-C showed ED₅₀ 0.1 μM. However, the conjugates I and II exhibited 287% and 238% of $\text{T}\text{C}$ at 50 mg/kg/day × 5 doses against L1210 leukemic mice, respectively, while ara-C at 25 mg and 50 mg/kg/day × 5 gave the respective 127% and 110% of $\text{T}\text{C}$.     

III. Possible mechanisms involved in the presynaptic cholinotoxicity due to ethylcholine aziridinium (AF64A) invivo

AF64A is a toxin which can diminish irreversibly cholinergic transmission $\text{in}$$\text{vivo}$ (1, 2). Disruption of neurotransmitter function $\text{in}$$\text{vivo}$ is specific to the cholinergic system when AF64A is administered in nanomolar quantities (3, 4). The mechanisms involved appear to be mediated presynaptically (2). The neurochemical and behavioral consequences of AF64A administration are reminiscent of similar measures in patients with Alzheimer's disease (5,6). Consequently, we have suggested tentatively that the AF64A treated animal may be explored as a potential animal model of this debilitating disease state (7). In this report we provide a brief overview of our recent findings using this compound $\text{in}$$\text{vivo}$, attempt to correlate these findings with those of others with similar aziridinium agents $\text{in}$$\text{vitro}$, and propose a possible mechanism of action of AF64A $\text{in}$$\text{vivo}$, based on recent observations made in our laboratories.     

Synthesis and antitumor activity of 1-β-D-arabinofuranosylcytosine-5′-diphosphate-prednisolone and -cortisol

Superior antitumor activity and reduced toxicity of 1-β-arabinofuranosylcytosine (ara-C) conjugates of corticosteroids through a phosphodiester linkage have prompted us to synthesize the new ara-C conjugates of corticosteroids through a pyrophosphate diester linkage. Condensation of ara-CMP morpholidate with the steroid-21-monophosphates in pyridine at room temperature for 6 days and the subsequent separation on a DE-52 (formate) column using a linear gradient of 0?0.5 M triethylammonium formate (pH 7.0) gave ara-CDP-prednisolone (I) and ara-CDP-cortisol (II) in 37–55% yield. The conjugates were hydrolyzed to ara-CMP and the corresponding steroid-21-monophosphate by phosphodiesterase I. Conjugates I and II inhibited the $\text{in}\text{,}\text{vitro}$ growth of L1210 lymphoid leukemia by 50% (ED₅₀) at 0.03 and 0.08 μM, respectively, while ara-C and ara-CMP showed the respective ED₅₀ of 0.1 μM and 0.05 μM. Conjugates I and II exhibited ILS values of 116 and 86%, respectively, at 40 mg (53.7 μmole)/kg/day × 5 against L1210 leukemic mice, while that of ara-C at the nearly same dose (49 μmole/kg/day × 5) was 65%.     

Further evidence for the involvement of D2, but not D1 dopamine receptors in dopaminergic control of striatal cholinergic transmission

The relative involvement of D₁ (cyclase linked) and D₂ dopamine receptors in dopaminergic control of striatal cholinergic transmission has been investigated in the rat by comparing the effects of SKF 38393 and LY 141865 (which act as specific agonists at D₁ and D₂ dopamine receptors, respectively) on striatal acetylcholine and dopamine metabolite concentrations and on the potassium-evoked release of ³H-acetylcholine from rat striatal slices. LY 141865 given systemically produced a dose-dependent increase in acetylcholine concentrations and a concomitant reduction of homovanillic and dihydroxyphenylacetic acid levels in the striatum (ED₅₀ 0.1 mg/kg) whereas SKF 38393 (1–30 mg/kg) did not. SKF 38393 (30 mg/kg) also failed to modify the LY 141865 (1 mg/kg) induced alterations of striatal acetylcholine and dopamine metabolite levels when given concomitantly with the latter compound. In experiments $\text{in vitro}$, LY 141865 reduced (EC₅₀ 0.14 μM), whereas SKF 38393 (up to 100 μM) failed to affect, the potassium-evoked release of ³H-acetylcholine from striatal slices. When given concomitantly with LY 141865, SKF 38393 (10 μM) did not modify the ability of the former compound to diminish striatal ³H-acetylcholine release. Finally, SKF 38393 also failed to affect the release of striatal ³H-acetylcholine after chemical lesion of the nigro-striatal dopaminergic pathway. The present results provide evidence for the involvement of D₂ but not D₁ dopamine receptors in dopaminergic control of striatal cholinergic transmission and indicate that D₁ dopamine receptors do not exert any modulatory influence on D₂ dopamine receptor mediated dopaminergic transmission.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

dl-N-methyl-3-(o-methoxyphenoxy)-3-phenylpropylamine hydrochloride, Lilly 94939, a potent inhibitor for uptake of norepinephrine into rat brain synaptosomes and heart.

dl-N-Methyl-3-(o-methoxyphenoxy)-3-phenylpropylamine hydrochloride, Lilly 94949, is a potent inhibitor for uptake of norepinephrine (NE) into synaptosomes of rat brain with inhibitor constant (Ki) value of 1.8 × 10⁻⁷M. Lilly 94939 profoundly reduces the $\text{in vivo}$ accumulation of radioactivity from labeled NE in heart with ED₅₀ value of 1.5 mg/kg i.p. The inhibitory effects of the compound in synaptosomes and heart are most profound within 15 min of an intraperitoneal injection of Lilly 94939 at 10 mg/kg but much deminished at the 4th hr. These properties are in great contrast with its trifluoromethyl analog, Lilly 110140, which has previously been reported as a selective inhibitor of serotonin uptake in synaptosomes and without any effect on the accumulation of radioactivity from labeled NE in heart.     

Aminoglycoside-Ca++ interactions in skeletal muscle preparations.

Neomycin and streptomycin are equally potent in inhibiting rates of Ca uptake by skeletal muscle fragmented sarcoplasmic reticulum (FSR) and also by heavy FSR isolated between 2000–8000 × G, and affect both membrane preparations to the same extent. In media containing initial free Ca concentrations of 10⁻⁷ and 10⁻⁶M (pCa 7, 6) the inhibition by neomycin of FSR Ca uptake rate is dependent upon the pCa and appears to be competitive, the antibiotic concentrations producing half-maximal effects on Ca uptake being 0.075mM at pCa and 0.53mM at pCa 6. Both MgATPase and CaATPase of FSR are inhibited by neomycin but Ca efflux from FSR partially loaded with Ca oxalate is increased by the antibiotic. Neomycin is 3.2 times as potent as streptomycin in producing 50% neuromuscular blockade in rat phrenic nerve-diaphragm preparations. The ED₅₀ for blockade by neomycin is 0.23mM in medium containing 1.3mM total Ca, but 0.99mM neomycin is required when the medium total Ca is raised to 2.6mM, although the dose-response curves are parallel. Minimal penetration of neomycin into the muscle cell and undetectable effect on the sarcoplasmic reticulum $\text{in}$$\text{situ}$ is indicated by the small decrease in contractility and unchanged relaxation time after exposure to 4.88mM antibiotic, i.e. 20 times the ED₅₀ for neuromuscular blockade.     

On the mechanism of tolerance to isoproterenol-induced accumulation of cAMP in rat pineal in vivo.

Less cyclic adenosine 3′:5′ monophosphate (cAMP) accumulated in rat pineal gland, $\text{in}$$\text{vivo}$, after two doses of l-isoproterenol (5mg/kg, i.p.) than after one dose. A single injection of l-isoproterenol decreased the ability of l-isoproterenol to activate adenylate cyclase and increased the activity of the low Km phosphodiesterase (PDE). Tolerance to l-isoproterenol-induced accumulation of cAMP in rat pineal $\text{in}$$\text{vivo}$ may be due to decreased responsiveness of adenylate cyclase as well as to increased activity of PDE.     

Evidence that opiate receptors mediate suppression of hypertonic saline-induced drinking in the mouse by narcotic antagonists

The effects of naloxone, its $\text{dextro}$-stereisomer, and five other narcotic antagonists were determined on water intake induced by intracellular dehydration in the mouse. The intraperitoneal administration of a 2M sodium chloride solution served as the model for intracellular dehydration. $\text{1}$-Naloxone (0.01-10 mg/kg) reduced drinking in a dose-dependent fashion with an ED₅₀ of 0.55 mg/kg. In contrast, $\text{d}$-naloxone failed to suppress water consumption at doses up to 10 mg/kg. The other narcotic antagonists tested --- naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine --- also produced dose-dependent decreases in water consumption. The order of potency of these narcotic antagonists in suppressing water intake was highly correlated with their orders of potency in other procedures involving the opiate receptor. The stereoselectivity and order of potency suggest that the suppressant effects of the narcotic antagonists on drinking induced by hypertonic saline administration in the mouse are mediated through an opiate receptor-dependent mechanism.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

Inhibition of platelet thromboxane A2 synthase activity by sodium 5-(3′-pyridinylmethyl)benzofuran-2-carboxylate

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinymethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets $\text{in}$$\text{vitro}$, as well as in rhesus monkey platelets $\text{ex vivo}$. A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI₂ biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 at 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation,. Similar effects were obtained by oral administration of 1–5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxgenase inhibitors and equal to PGI₂ in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cylooxygenase inhibitors suggesting that its efficacy dependened in part of endogenous PGI₂ formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of patho[hysiological states.     

The significance of physiological [Ca2+] and [Mg2+] for in vitro experiments on synaptic transmission

Since 1932 $\text{in}$$\text{vitro}$ physiological and pharmacological studies on neuromuscular and other types of synaptic transmission have been carried out usually in Krebs' of similar balanced electrolyte solutions. It has been disregarded, however, that although the total calcium [Ca_t] (2.5 mM) and [Mg_t] (1.2 mM), are about the same in human plasma and in Krebs' solution, the physiologically important [Ca²⁺] and [Mg²⁺], primarily because of binding to plasma proteins, are much lower in plasma (1.1 and 0.6 mM) than in Krebs' solution (2.0 and 1.1 mM). We observed that in a modified Krebs' solution in which the [Ca_t] and [Mg_t] are 1.4 and 0.9 mM respectively and the [Ca²⁺] and [Mg²⁺] are about the same as in human plasma, the Ca²⁺ dependent volley output of acetylcholine is less and the inhibition of the electrically induced isometric twitch tension of the rat phrenic nerve - hemidiaphragm preparation by nondepolarizing neuromuscular blocking agents and certain antibiotics is greater than in conventional Krebs' solution, in which the [Ca²⁺] and [Mg²⁺] are higher than $\text{in}$$\text{vivo}$. Similarly, during electrical field stimulation of the guinea-pig myenteric plexus - longitudinal muscle preparation volley output of acetylcholine is lower and the inhibition of the isometric contraction of the muscle by normophine is greater in modified than in conventional Krebs' solution. It is suggested that for greater relevance to $\text{in}$$\text{vivo}$ conditions the [Ca²⁺] and [Mg²⁺] of balanced electrolyte solutions used in in vitro experiments on synaptic transmission should be the same as in human plasma or in the plasma of the species of the experimental animal.     

Substance P found to lower body temperature and aggression.

Synthetic substance P (SP) was bioassayed in mice by a procedure comprising eleven subtests. Two replications of a dose-response study were conducted at the time of the peak effect of the intravenous injection of SP. Single doses of 31 and 63 ng/kg significantly decreased body temperature. SP, [D-Arg¹]-SP, and [des-NH₂-Arg¹]-SP comparably lowered blood pressure, but [D-Arg¹]-SP and [des-NH₂-Arg¹]-SP were $\text{1}\text{20}$ to $\text{1}\text{10}$ as active as SP in lowering body temperature. The activities that lower body temperature and blood pressure may be different. The thyrotropin and luteinizing hormone releasing hormones (TRH and LH-RH) did not lower body temperature. SP also decreased the aggressive response (ED₅₀, 89 ng/kg).     

Increase in rat striatal acetylcholine content by bromocriptine: Evidence for an indirect dopaminergic action

Bromocriptine, at the optimal dose and time of 4 mg/kg, 90 min, increased the content of acetylcholine in the rat striatum by about 30% without affecting the acetylcholine content in other brain regions. Striatal choline acetyltransferase and acetylcholinesterase activities and sodium-dependent high affinity choline uptake were not affected by the $\text{in vivo}$ administration or the $\text{in vitro}$ incubation with even high amounts of the drug. The increase in striatal acetylcholine by bromocriptine was mediated through the dopaminergic system since pretreatment with pimozide or penfluridol, powerful dopamine receptor antagonists, completely prevented the effect while parachlorophenylaline and phenoxybenzene pretreatment were ineffective. The action of bromocriptine, differently from that of apomorphine, was also blocked upon inhibition of tyrosine hydroxylase by alphamethylparatyrosine, suggesting that intact catecholamine synthesis is necessary for the drug to act. The requirement of dopamine by bromocriptine was further indicated when no potentiation of the cholinergic response to bromocriptine occurred following induction of dopamine receptor supersensitivity by long-term 6-hydroxydopamine lesion of the nigroneostriatal pathway. On the other hand, evidence is presented to show that bromocriptine acts in synergism with dopamine as the latency period for the onset of bromocriptine's cholinergic action was significantly decreased when it was administered in combination with a subthreshold dose of L-dopa, the dopamine precursor. There also was no summation of bromocriptine's increase with apomorphine's increase in striatal acetylcholine content at supramaximal doses possibly indicating that the same population of intrastriatal cholinergic neurons is the common target of both drugs.It is proposed that bromocriptine exerts an inhibitory effect on the striatal cholinergic neurons through a stimulation of the dopaminergic system but, differently from apomorphine, it requires the presence of endogenous dopamine for its action.     

The effects of narcotic analgesics and narcotic antagonists on ganglionic transmission in the rat.

The effects of narcotic analgesics, narcotic-antagonist analgesics and narcotic antagonists on ganglionic transmission in the superior cervical ganglia of the rat were studied $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$ administration of morphine, meperidine, methadone, pentazocine or naltrexone blocked ganglionic transmission. Levorphanol, cyclazocine, nalorphine and naloxone had no effect on ganglionic transmission in this procedure. $\text{In}$$\text{vitro}$ studies confirmed the $\text{in}$$\text{vivo}$ results with the exception of levorphanol, cyclazocine and nalorphine, which were also found to block ganglionic transmission $\text{in}$$\text{vitro}$. In both preparations, naloxone did not antagonize the effect of morphine, suggesting that the effects of morphine and the other opiates were nonspecific. Similar potency of $\text{d}$- and $\text{l}$-isomers of pentazocine and cyclazocine support this conclusion. The observation that naltrexone blocked ganglionic transmission, but the other pure narcotic antagonist, naloxone, was inactive is somewhat unique to this test procedure and possibly significant.     

Synthesis of porcine leumorphin and some of its biological activities

The carboxy-terminal nonacosapeptide sequence of porcine preproenkephalin B contains the sequence of Leu-enkephalin at its amino terminus. The endogenous existence of this peptide, leumorphin, has not yet been proved. Synthesis of leumorphin was carried out by a solid-phase technique and the purity and structure of the synthetic peptide were confirmed. Synthetic porcine leumorphin exhibited a dose-dependent opiate effect (ED₅₀ 4.70 · 10^?9 M) on electrically stimulated contraction of the guinea pig ileum preparation. The potency was about 100 times as high as that of Leu-enkephalin. Leumorphin was less potent than dynorphin(1–13) (ED₅₀ 0.38 · 10^?9 M) but it was more active than $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{endorphin}$ (ED₅₀ 18 · 10^?9 M). The opiate activity was only partially reversed by naloxone. Intracisternal injection of synthetic leumorphin caused significant analgesia in mice (ED₅₀ 7.31 nmol/mouse). The potency was lower than that of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{endorphin}$ (ED₅₀ 0.60 nmol/mouse) but higher than that of dynorphin(1–13) (ED₅₀ 16.10 nmol/mouse). Intracisternally injected leumorphin did not produce such a violent behavioral effect as did dynorphin(1–13), and it exhibited a mild sedative effect. The data supports the concept that leumorphin is a new type of opioid peptide and that the synthetic preparation will be useful for further biological and immunological studies on this peptide.     

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 $\text{in vivo}$.     

Effects of atropine treatment on cortical,striatal and hippocampal high-affinity uptake of choline in the mouse

Changes in high-affinity uptake of choline (H.A._Ch) were studied in synaptosomes from different mouse brain regions following intravenous (i.v.) administration of atropine (0.3–30 mg/kg body weight) $\text{in vivo}$. The Ch-uptake was expressed as a Ch-uptake index, defined as the ratio between H.A._Ch and the corresponding choline acetylt-ransferase (ChAt) activity. The Ch-uptake index was highest in the hippocampus and lowest in the striatum. In the hippocampus a dose-dependent increase in this index was found following atropine treatment, while the striatal Ch-uptake index was unaffected by atropine. Atropine given i.v. in a dose of 10 mg/kg induced a 86% increase in V_max in synaptosomes from the hippocampus.     

Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

³H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the $\text{Torpedo}$ fish. The K_d for binding to intact and solubilized rat brain membranes was 5.6 × 10^?9 M and 1.1 × 10^?8M respectively, and the binding capacity 2.0 × 10^?14 and 3.0 × 10^?13 moles /mg protein respectively. The K_d for Torpedo membranes was 3.1 × 10^?7M and the binding capacity 6.8 × 10^?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in $\text{Torpedo}$ membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with ³H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.