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.     

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 $\text{T}\text{C}$.     

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.     

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).     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

Peptide receptors in human lung tumor cells in culture: vasoactive intestinal peptide (VIP) and secretin interaction with the Calu-1 and SW-900 cell lines

We have investigated the interaction of VIP and secretin with two human lung carcinoma cell lines in cultures, SW-900 and Calu-1. ¹²⁵I-labeled VIP binds to and is inactivated by SW-900 and Calu-1 cells in a time- and temperature-dependent manner. The rates of binding and of inactivation were higher at 30°C than at 15°C. At equilibrium, native VIP competitively inhibited the binding of ¹²⁵I-VIP in the 10^?10?10^?7M range, half-maximal inhibition being observed at 1.2 nM in SW-900 cells and at 1.1 nM VIP in Calu-1 cells. Scatchard analysis indicated two classes of binding sites with similar characteristics in both cell lines. SW-900 cells have 27 600 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.34}\text{nM}$) and 1062 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 61.4}\text{nM}$). Calu-1 cells have 36 300 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.33}\text{nM}$) and 1148 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 78.6}\text{nM}$). Secretin inhibited tracer binding but with a 5000 times lower potency than native VIP in both cell lines.     

The effect of antiestrogens on the nuclear binding of the estrogen receptor

Experiments were designed to determine whether or not various antiestrogens in direct competition with estradiol-17β (E₂) would inhibit the translocation of the estrogen receptor complex from the cytoplasm to nuclei in rat uterine tissue. Incubation of the antiestrogens CI-628, $\text{cis}$-clomiphene, U-11,100A and MER-25 with rat uteri caused the nuclear uptake of the antiestrogen receptor complex which was greatest for most antiestrogens at concentrations of 1 × 10^?6 to 1 × 10^?5M. At higher concentrations of CI-628, $\text{cis}$-clomiphene, and U-11,100A the nuclear binding of the antiestrogen receptor complex was greatly decreased. Incubation of the antiestrogens with E₂ resulted in a dramatic inhibition of the nuclear uptake of the estrogen receptor. $\text{Trans}$-clomiphene, a weak estrogen, did not inhibit the movement of the uterine cytoplasmic receptor into the nuclear fraction.     

Mode of action of sulfoxides in preventing loss of activity of 11β-hydroxylase in cultured bovine adrenocortical cells

Previously, loss of 11β-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me₂SO) were included in the medium. In the present experiments, we tested the hypothesis that Me₂SO protects 11β-hydroxylase by scavenging OH^? radicals. Substances known to react with OH^? at high rates and non-toxic enough to be used at concentrations of 10–100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11β-hydroxylase in the presence of 50 μM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me₂SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH^? radicals do not appear to be involved in loss of 11β-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11β-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to $\text{1}\text{200}$ of that required for Me₂SO. Previously, we have demonstrated that 11β-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11β-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11β-hydroxylase. Me₂SO showed an ED₅₀ for inhibition of 11β-hydroxylase activity of > 1 M, in contrast to its ED₅₀ for protection of 34 mM. For metyrapone, however, the ED₅₀ for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED₅₀-values for inhibition of 11β-hydroxylase that were substantially higher than the ED₅₀-values for protection. Sulfoxides may have a mixed mode of action in protection of 11β-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.     

5HT2 receptors in the rat portal vein: Desensitization following cumulative serotonin addition

Contractile responses to serotonin were examined $\text{in}$$\text{vitro}$ in the longitudinal portal vein to determine whether such responses were mediated by the interaction of serotonin with 5HT₁ receptors (those that preferentially bind [³H]serotonin) or 5HT₂ receptors (those that preferentially bind [³H]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the affinity for serotonin receptors in the rat portal vein and the ability to bind to 5HT₂, but no 5HT₁ receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED₅₀ value) relative to contractions produced by the non-cumulative addition of serotonin. Affinities of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentration-response curves compared to affinities obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT₂ receptor affinities of antagonists in the rat portal vein are best determined by the shift of non-cumulative responses to serotonin.     

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.     

The dextro and levorotatory isomers of N-phenylisopropyladenosine: stereospecific effects on cyclic AMP-formation and evoked synaptic responses in brain slices.

The stereoisomers of N⁶-phenylisopropyladenosine elicit accumulations of cyclic AMP in brain slices via interaction with adenosine-receptors. The response in guinea pig cerebral cortical slices and in rat hippocampal slices is blocked by theophylline and potentiated by biogenic amines. A chelator, EGTA, potentiates the response to phenylisopropyladenosine in guinea pig cerebral cortical slices. The $\text{1}$-isomer (EC₅₀ 25 μM) is four- to five-fold more potent than the $\text{d}$-isomer in eliciting accumulations of cyclic AMP in brain slices. In a rat coronal hippocampal slice $\text{in vitro}$, $\text{1}$-phenylisopropyladenosine (IC₅₀ ～ 0.7 μM) reduces the amplitude of evoked synaptic responses generated $\text{via}$ a monosynaptic pathway to the CA1 pyramidal neurons. The $\text{d}$-isomer is nearly one hundred-fold less potent. Thus, the adenosine-receptors involved in the electrophysical response appear much more stereoselective for the $\text{1}$-isomer of phenylisopropyladenosine than the adenosine-receptors involved in cyclic AMP-generation in brain slices.     

Peptide hormone degradation by a rat mast cell chymase-heparin complex

Material released from rat mast cells by compound $\text{48}\text{80}$ gave parallel responses using ACTH and β-endorphin radioimmunoassays. However, incubation of these labeled compounds under conditions of radioimmunoassay with released material and chromatography on Sephadex G-25 provided evidence that neither ACTH nor β-endorphin were present in the material released from mast cells, but represented an artifact produced by the presence of a protease. Analysis of the released enzyme on Sephadex G-75 under non-dissociative conditions yielded an active enzyme complex with a M_r > 150,000. Under dissociative conditions, the M_r of the enzyme was 25,000. The dissociated enzyme reassociated with purified rat mast cell heparin to form the high molecular weight complex. Further investigation of pH, substrate and inhibitor specificity showed that the peptide degradation is due to a chymotrypsin-like protease, the previously described mast cell chymase, which is active in degrading β-endorphin, ACTH, and ACTH^1–24.     

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.     

3β-Hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal,testis and ovary during late gestation

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ (3β-HSDH) was measured in the rhesus monkey ($\text{Macaca mulatta}$) placenta, fetal adrenal (whole organ minus medulla), testis and ovary during late gestation (Days 145–162). Activities were evaluated from the conversion of [³H]-pregnenolone to [³H]progesterone. The maximum enzyme velocity (V_m) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg^?1protein) than in microsomes from the other tissues. Testicular V_m was greater than either ovarian or placental V_m which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg^?1protein, respectively). Apparent Michaelis-Menten constants in the adrenal, placenta, testis and ovary averaged 1.8,2.5,0.27 and 0.16 μM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 μM (adrenal), 2.1 μM (placenta), 0.74 μM (testis) and 0.13 μM (ovary). 3β-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes.Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 3β-HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

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}$.     

Inhibition of norepinephrine N-methyltransferase by 1-aminoindans,conformationally rigid analogs of benzylamines

Bicyclic analogs of benzylamine (with the α carbon connected by one or more methylene units to the ortho position of the benzene ring) inhibited rabbit adrenal norepinephrine N-methyltransferase (NMT) $\text{in}$$\text{vitro}$. Inhibition was greater when the second ring contained five carbons (1-aminoindan) than when it contained four, six, or seven carbons. Substitution of chlorines on the benzene ring further enhanced the inhibition by 1-aminoindan. The most active inhibitor, 4,5-dichloro- 1-aminoindan, showed competitive kinetics with ?-norepinephrine as the variable substrate, and the K_i for this compound as an inhibitor of adrenal NMT was 0.22 μM. 4,5-Dichloro-1-aminoindan significantly decreased epinephrine concentration in the adrenal glands of exercised rats, suggesting that it was effective in inhibiting NMT $\text{in}$$\text{vivo}$.     

Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

Cytosolic free calcium concentration and intracellular calcium distribution in lymphocytes from cystic fibrosis patients

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intercellular Ca²⁺ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca²⁺ ($\text{98 ± 5}\text{vs}\text{102 ± 7}\text{nM}$), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca²⁺ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca²⁺ is about 25% higher in CF lymphocytes than in control. Of this excess Ca²⁺, about 50% appears to be sequested in mitochondria. This suggests that some difference in Ca²⁺ handling does exist, but the significance of this cystic fibrosis remains to be determined.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.