Morphine dependence in the isolated guinea-pig ileum and its modification by p-chlorophenylalanine.

Experiments were performed to quantitatively determine morphine physical dependence in the isolated guinea-pig ileum and to assess the influence of p-chlorophenylalanine (PCPA) on its development. Ileum segments taken from animals treated with 10 s.c. injections of 100 mg/kg of morphine, given at intervals of 8 hr without interruption, responded with intense, prolonged, dose-dependent contractions to the $\text{in}$$\text{vitro}$ administration of naloxone, although contractions guinea-pigs also responded to naloxone, although contractions were smaller and of short duration. The sensitivity to naloxone on segments isolated from morphinized animals was compared to that of controls. Ilea from morphine-treated guinea-pigs were 8 to 32 times more sensitive to naloxone, as determined by a shift in the naloxone concentration-response curve to the left. There was also a three-fold increase in the maximum response. This phenomenon was taken as evidence of narcotic dependence. PCPA, given before morphine administration, at doses producing only a slight (11%) decrease in intestinal serotonin (5-HT) levels, partially reduced the sensitivity of the morphine-treated ileum to naloxone. However, high doses of PCPA, decreasing intestinal 5-HT by 40%, enhanced the abstinence-like effects of naloxone in the morphine pretreated ileum. PCPA by itself changed the responsiveness of the non-morphinized ileum to naloxone. The direction and magnitude of the change produced by PCPA alone was roughly equivalent to that produced by the serotonin depletor in the morphinized ileum. This finding indicates that PCPA has no effect upon the development of physical dependence in the isolated ileum. It remains to be determined whether or not the increased sensitivity to naloxone induced by high doses of PCPA has something in common with the changes in responsiveness to the antagonist induced by narcotics.     

Early exposure to naloxone increases blood pressure in normotensive and hypertensive rats

Continuous $\text{in}\text{utero}$ and postpartum exposure of SH and WKY rats to naloxone results in a significant increase in their systolic blood pressure relative to respective control animals. After six weeks of age, however, naloxone was no longer effective in sustaining this increase in blood pressure. Chronic exposure to naloxone beginning at three weeks of age failed to produce any significant differences in blood pressure between treated and control animals. Although naloxone has been shown to elevate blood pressure in hypotensive states, this report represents the first example of an increase produced by the narcotic antagonist in the normotensive state.     

Humoral endorphin: A new endogenous factor with opiate-like activity

Humoral (H) endorphin, a novel endogenous opioid ligand detected in brain, blood and cerebrospinal fluid was tested in a series of opiate sensitive assays. H-endorphin displaced radiolabeled enkephalin from its specific bindings sites and inhibited the electrically evoked contraction of the guinea pig ileum and mouse vas deferens. When injected to unanesthesized animals, humoral endorphin induced analgesia in rats and mydriasis in mice. The activity of H-endorphin both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$ attests to its opioid nature. However, while its antinociceptive effect was blocked by naloxone, mydriasis induced by H-endorphin was resistant to the effect of the opiate antagonist. Similarly, intermediate concentrations of naloxone inhibited the effect of H-endorphin on the guinea pig ileum while its effect on the mouse vas deferens was completely refractory to naloxone. The physiological function of humoral endorphin in various naturally occuring states that show similar paradoxical interactions with naloxone is discussed.     

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.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Intracellular Na+ and K+ shifts induced by contractile activities of rat skeletal muscles

The effects of direct and indirect electrical stimulation on intracellular potassium and sodium contents ([K]_i and [Na]_i, respectively) in rat soleus muscle (SOL) and extensor digitorum longus muscle (EDL) were investigated under in vivo conditions. The changes of [K]_i and [Na]_i contents in both muscles which were stimulated indirectly reached respective values at 30 min or 1 hr after the beginning of stimulation, whereas those of EDL stimulated with 60 Hz changed gradually through 2 hr stimulation. The shifts of [K]_i and [Na]_i in EDL occurred during the twitch contraction at considerably lower frequency stimulation (0.5–10 Hz), whereas those in SOL were observed during the tetanus contraction at high frequency stimulation (10–40 Hz). The difference of change in cationic shifts between EDL and SOL under low frequency stimulation was reduced by ouabain treatment, though the difference was still significant. When the muscles were indirectly stimulated 6000 times at 1,5,10 and 20 Hz, the cationic shifts in EDL were greater than those in SOL, extending over all frequencies. It was concluded that such a difference in ionic shift between contracting EDL and SOL may be primarily due to the difference in unidirectional ionic fluxes per stimulation and, secondly, to the difference in Na⁺-K⁺ pump activity.     

Effects of morphine on dopamine-stimulated adenylate cyclase and on cyclic GMP formation in primate brain amygdaloid nucleus.

In homogenates of $\text{Macaca}$$\text{mulatta}$ (Rhesus) or $\text{Cebus}$$\text{apella}$ amygdaloid nuclear complex, adenylate cyclase activity was approximately doubled by either 10μ$\text{M}$ dopamine or 8m$\text{M}$ NaF. In the presence of morphine, the stimulation by dopamine was reduced. A 90–100% inhibition of the dopamine stimulation was obtained with 20μ$\text{M}$, and a 50% inhibition, with 5μ$\text{M}$ morphine. The effects of 10μ$\text{M}$ morphine on dopamine stimulation were reversed by 10μ$\text{M}$ naloxone. Morphine itself did not significantly affect the basal adenylate cyclase activity, but in the presence of 10μ$\text{M}$ morphine the stimulation by 8m$\text{M}$ NaF was reduced approxiamtely 50%. The data suggest an action of morphine at a receptor site which is distinct from the dopamine receptor, but which inhibits the dopamine-stimulated adenylate cyclase. In addition, the cyclic GMP content of $\text{Cebus}$ amygdala slices was reduced by 50–75% during incubation for 5–20 minutes with morphine. Maximum effects on cyclic GMP were obtained with 10μ$\text{M}$, and half-maximum effects, with 0.1μ$\text{M}$ morphine. The effect of morphine on amygdala cyclic GMP was not reversed by naloxone. Thus, this action of morphine may not be receptor mediated, or may involve the interaction of morphine with receptors other than the opiate receptor.     

Dopamine and norepinephrine stimulate somatostatin release by median eminence fragments invitro

The effect of catecholamines on somatostatin release by median eminence (ME) fragments was evaluated using an $\text{in}$$\text{vitro}$ incubation system. Adult male rats were used as tissue donors. Somatostatin release was readily detected during short-term incubations (10 and 30 minutes). Dopamine (DA) significantly stimulated somatostatin release during a 30 minute incubation period at the two doses tested (0.6 and 6 μM). Under similar conditions, norepinephrine (NE) stimulated somatostatin release only at the 6 μM dose. Using a shorter incubation period (10 min) and a 6 μM dose, only DA stimulated somatostatin release. The effects of DA and NE were specifically blocked by the $\text{in}$$\text{vitro}$ addition of pimozide or phentolamine, respectively, suggesting that dopaminergic and noradrenergic receptors may be present in the somatostatinergic terminals of the ME. The results indicate that both DA and NE may be involved in the regulation of somatostatin secretion.     

Precocious development in utero of certain UDP-glucuronyltransferase activities in rat fetuses exposed to glucocorticoids.

The first precocious development of UDP glucuronyltransferase in the mammalian fetus $\text{in utero}$ by a known compound of endogenous origin is described. Intraperitoneal injection of cortisol (8 mg) into maternal rats on days 14 and 15 of gestation stimulated fetal-liver transferase activity from near zero to $\text{1}\text{2}$ maternal levels by day 17; 0.3 mg dexamethasone, possessing a longer biological half-life, raised activity to full maternal level by day 16. In controls, injected with solvent only, fetal-liver transferase remained low on day 16. With both glucocorticoids, transferase stimulation was dose-dependent. Transferase activities were assayed in a range of digitonin concentrations from zero to above optimal for enzyme activation. Activities stimulated were towards $\text{o}$-aminophenol, $\text{p}$-nitrophenol, 1-naphthol and serotonin. Activities towards bilirubin, morphine and testosterone were not stimulated. The former group of activities are stimulated by glucocorticoids in culture and normally reach approximate adult levels just before birth; the latter group are not so stimulated on culture and normally reach adult levels after birth. Implications of these findings are discussed.     

Stimulation by cyclic nucleotides of prostaglandin E production in isolated Graafian follicles

Rat Graafian follicles isolated intact responded to 8-Br-cyclic AMP and 8-Br-cyclic GMP with increased prostaglandin E (PGE) production during a 6 h incubation. By contrast, 8-Br-cyclic IMP, 8-Br-5′ AMP and 8-Br-5′ GMP were inactive in this respect. The effect of 8-Br-cyclic AMP and 8-Br-cyclic GMP was noted only after a lag period of about 4 h. Choleragen, LH, and the phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine; IBMX) also stimulated PGE production. Actinomycin D and cycloheximide given simultaneously with 8-Br-cyclic AMP or LH prevented the stimulatory effect of these agents. Concomitant addition of arachidonic acid did not overcome the effect of these inhibitors.Administration of hCG $\text{in vivo}$ or incubation with LH $\text{in vitro}$ did not elevate endogenous ovarian free arachidonate, while PGE production was enhanced. Dexamethasone prevented this stimulatory effect of hCG.Collectively, the results suggest that stimulation of ovarian PGE production by cyclic mucleotides and LH is dependent on $\text{de novo}$ synthesis of one more components of the PG synthetase systme rather than on substrate availability. Cyclic nucleotides may mediate the stimulatory effect of gonadotropins on PGE production     

Role of calcium ions in the stimulatory actions of luteinizing hormone in isolated ovarian cells: Studies with divalent-cation ionophores

The divalent-cation ionophores A23187 and ionomycin exert dose-dependent suppressive effects on the stimulatory actions of luteinizing hormone in ovarian cells $\text{in}\text{vitro}$. Micromolar concentrations of both A23187 and ionomycin can inhibit the production of progesterone and the stimulation of ornithine decarboxylase activity. Inhibitory concentrations of these ionophores deplete total cell content of calcium, and also seem to suppress protein synthesis.     

Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Modulation by the ratio S-adenosylmethionineS-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. $\text{S-}\text{Adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio $\text{S-}\text{adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ can modulate phosphorylation of a specific protein.     

Evidence for morphine and morphine-like alkaloid responses resistant to naloxone blockade in the rat vas deferens.

The application of morphine or surrogates to the isolated rat vas deferens maintained at 37° C in Tyrode solution, produced an increase in the electrically induced muscular twitch. In contrast, leucine enkephalin or D-alanine₂methionine enkephalinamide produced a dose-dependent inhibition of the muscular twitch. The effect of morphine and derivatives was not antagonized by naloxone, but the depression caused by the opiate pentapeptides or β-Endorphin was readily antagonized and reversed by naloxone. Tolerance developed to the $\text{in vitro}$ effect of morphine; vasa deferentia obtained from tolerant-dependent rats were about six times less sensitive to the effect of morphine and about five times less sensitive to the depression caused by leucine enkephalin as compared to their respective paired, placebo implanted control rats.     

Stimulation of intralysosomal proteolysis by cysteinyl-glycine,a product of the action of γ-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversible inhibitor of γ-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with $\text{l}\text{-(αS,5S)-α-}\text{amino}\text{-3-}\text{chloro}\text{-4,5-}\text{dihydro}\text{-5-}\text{isoxazoleacetic}$ acid (acivicin), an irreversible inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepated kidney membranes or purified γ-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of γ-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or γ-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of γ-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.     

Mechanism of development of tolerance to injected morphine by guinea pig ileum.

Injection of a large dose of morphine into a guinea pig results in a block of electrically-induced contractions of the ileum $\text{in vitro}$. A similar dose is almost ineffective in guinea pigs given morphine chronically. The time course for development of this tolerance has been determined in guinea pigs injected twice daily with morphine 100 mg/kg and challenged on various days with 750 mg/kg of the drug. Animals similarly injected but not challenged served as controls. The inhibitory effect of the challenging dose on electrical stimulation of longitudinal muscle decreased with successive days of morphine administration; by the 10th day there was almost complete tolerance to the challenging dose. Sensitivity of the tissues of chronically morphinized unchallenged controls towards acetylcholine, serotonin, histamine and norepinephrine was essentially the same as that of naive animals. The potency of morphine $\text{in vitro}$ in blocking electrical stimulation was also unchanged by chronic morphine administration in the above manner. Thus tolerance to injected morphine cannot be explained by reduced affinity of the drug for the opiate receptor. Tissues of chronically morphinized animals gave a contracture with naloxone, the extent of the contracture increasing with time of drug administration. This naloxone effect is attributed to displacement of morphine from a new opiate receptor site induced during morphine administration. It is suggested that this new receptor is involved in tolerance to injected morphine as well as some aspects of the withdrawal syndrome.     

Effect of N-ethylmaleimide on leucine transport in chang liver cells. I. Stimulatory effect on Na+-independent transport at low concentrations of leucine

Pretreatment of Chang liver cells with $\text{N-}\text{ethylmaleimide}$ (0.5 or 1 mM) stimulated Na⁺-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the uptake of leucine measured in Na⁺-replete medium was completely blocked by the addition of $\text{b-2-}\text{aminobicyclo}\text{[2,2,1]}\text{heptane}\text{-2-}\text{carboxylate}$ (5 mM), which shows that the L system participates in the stimulation. The Na⁺-dependent uptake of glycine was depressed by $\text{N-}\text{ethylmaleimide}$ pretreatment. The stimulation of the Na⁺-independent component of leucine uptake continued for at least 30 min after $\text{N-}\text{ethylmaleimide}$ treatment, while the inhibition of glycine uptake was progressive with time and the Na⁺-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with $\text{N-}\text{ethylmaleimide}$ is capable of increasing the Na⁺-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that $\text{N-}\text{ethylmaleimide}$ attacks the Na⁺-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.     

Inhibition of uterine contraction by synthetic parathyroid hormone fragment

The inhibitory effect of synthetic bovine parathyroid hormone fragment [bPTH-(1-34)] on rat uterine contraction was studied $\text{in vitro}$. Oxytocin, prostaglandin F_2α and acetylcholine produced log dose-related contraction. The addition of bPTH-(1-34) shifted the dose-response curves of the three agonists to the right. Two doses of bPtH-(1-34) were tested. The higher dose (400 ng/ml) caused a greater inhibition of the agonists than did the lower dose (40 ng/ml). bPTH-(1-34) also inhibited the uterine contraction elicited by electrical stimulation of the tissue. We suggest that bPTH-(1-34) has a non-specific depressing effect on the contractile mechanism of the uterine tissue.