Endogenous Opiate Receptors Research Articles

My Path to Psychiatry

Back to table of contents Previous article Why I Aspired to Be a PsychiatristFull AccessMy Path to PsychiatryJohn Krystal, M.D.John KrystalSearch for more papers by this author, M.D.Published Online:19 Oct 2017https://doi.org/10.1176/appi.pn.2017.10b1AbstractI feel lucky to be a psychiatrist. I came to this choice through a path in which I benefitted from incredible role models, mentors, colleagues, trainees, and patients who helped me grow as a person, doctor, and scientist.My first role model, and that of my brother Andrew (also a psychiatrist), was my father, Dr. Henry Krystal. He was a Holocaust survivor who turned experiences in the Nazi death camps into a psychiatric career focused on the problem of psychological trauma. Not only was his career inspiring, but he embodied traits and values—such as integrity, skepticism, curiosity, and humanism—that we aspired to emulate.In 1976, I entered the University of Chicago with only rough ideas about a career in medicine. There, Dr. Daniel X. Freedman was a charismatic psychiatric leader. I was interested in learning about addiction. Drs. Ed Senay, at Chicago, and Ed Khantzian, at Harvard, included me in activities at their methadone programs. I also interned with Dr. Martin Mitcheson at University College London, where I observed methadone, heroin, and even cocaine maintenance treatment. Seminal moments that shaped my career happened by accident. One epiphany came during a college biochemistry course with Dr. Earl Evans in which he discussed the discovery of endogenous opiates and opiate receptors, highlighting the work of Dr. Solomon Snyder and his colleagues. I realized that specific molecular mechanisms could be identified for addiction, and perhaps other psychiatric illnesses, and that there might be opportunities for transformative scientific discoveries. After that class, on the advice of Dr. Freedman, I contacted Dr. Richard Miller. I studied opiates and endorphins in his laboratory in my free time for the remainder of my college career. Another epiphany came in 1979, when Dr. Senay graciously brought me to the College on Problems of Drug Dependence meeting. There, I heard Dr. Mark Gold describe one of the first successes of translational neuroscience: the discovery of clonidine as a nonopiate treatment for opiate dependence. This work was a team effort at Yale University involving basic scientists, such as Drs. George Aghajanian and Eugene Redmond, and clinical investigators, such as Drs. Gold and Herbert Kleber. At that point, I wanted to go to Yale to join the hunt for molecular mechanisms of illness and novel treatments.I arrived at Yale for medical school in 1980 and I never left. Along the way, I had numerous influential mentors, particularly Drs. Dennis Charney, George Heninger, and Patricia Goldman-Rakic. Also, I have had wonderful colleagues and collaborators, whom I count among my most important influences and closest friends. I also have worked with enormously talented mentees, who forged successful independent careers. So far, my career has been more stimulating than I could have imagined. I focused my work on translational neuroscience and, with my colleagues, identified a promising new treatment, that is, the rapid antidepressant effects of ketamine. The opportunities to have an impact on mental illness through science are greater than ever. We are fortunate that despite some obvious challenges, this is the best time in the history of the field to pursue a career in academic psychiatry. ■John Krystal, M.D., is the Robert L. McNeil Jr. Professor of Translational Research and chair of the Department of Psychiatry at Yale University School of Medicine. He is also director of the NIAAA Center for the Translational Neuroscience of Alcoholism and director of the Clinical Neuroscience Division at the VA National Center for PTSD and serves on APA’s Council on Research. ISSUES NewArchived

Kappa-opioid receptors in the caudal nucleus tractus solitarius mediate 100 hz electroacupuncture-induced sleep activities in rats.

Previous results demonstrated that 10 Hz electroacupuncture (EA) of Anmian acupoints in rats during the dark period enhances slow wave sleep (SWS), which involves the induction of cholinergic activity in the caudal nucleus tractus solitarius (NTS) and subsequent activation of opioidergic neurons and μ-receptors. Studies have shown that different kinds of endogenous opiate peptides and receptors may mediate the consequences of EA with different frequencies. Herein, we further elucidated that high-frequency (100 Hz)-EA of Anmian enhanced SWS during the dark period but exhibited no direct effect on rapid eye movement (REM) sleep. High-frequency EA-induced SWS enhancement was dose-dependently blocked by microinjection of naloxone or κ-receptor antagonist (nor-binaltorphimine) into the caudal NTS, but was affected neither by μ- (naloxonazine) nor δ-receptor antagonists (natatrindole), suggesting the role of NTS κ-receptors in the high-frequency EA-induced SWS enhancement. Current and previous results depict the opioid mechanisms of EA-induced sleep.

Medial forebrain stimulation enhances intracranial nociception and attenuates morphine analgesia suggesting the existence of an endogenous opioid antagonist

The purpose of this experiment was to test in the rat the hypotheses that activation of the brain reward system would attenuate the effects of intracranial nociceptive stimulation and would potentiate the antinociceptive effects of morphine. In this experiment pain (nociception) was generated by electrical stimulation of a brain pain pathway, the mesencephalic reticular formation (MRF) of the rat. Reward pathway stimulation was to the medial forebrain bundle at the level of the lateral hypothalamus (MFB-LH). Current thresholds for escape from MRF stimulation were determined using a modification of the psychophysical methods of limits. MRF stimulation was delivered concurrently with different intensities of non-contingent MFB-LH stimulation. The effects of morphine and saline were determined under all stimulation conditions. Contrary to expectation MFB-LH stimulation significantly lowered MRF stimulation escape thresholds. Morphine administration elevated MRF thresholds in the absence of MFB-LH stimulation. However, this effect was blocked by concurrent MFB-LH stimulation. These findings, which mimic the effects of the opiate antagonist naloxone, i.e., potentiating of pain and antagonism of morphine's analgesic effects, suggest the presence of an endogenous opiate receptor antagonist.

A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels.

The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 +/- 6.8 % 5-HT-mediated, but not GTP-gamma-S-induced, inhibition of Ca(2+) current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP-gamma-S. 5-HT(1A) receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT(1A) receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca(2+) channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT(1A) receptors, with only a 13.4 +/- 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT(1A) receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-5-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT(1A) receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca(2+) channel.

Peptide transport system-1 (PTS-1) for Tyr-MIF-1 and Met-enkephalin differs from the receptors for either

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2) and Met-enkephalin share a saturable transport system (peptide transport system-1, PTS-1) across the blood–brain barrier but do not readily bind to each other's receptors. This information allows the unique opportunity to differentiate the transport protein(s) from the receptors for either peptide in brain endothelial cells. PTS-1 was studied in vitro by allowing radiolabeled Tyr-MIF-1 ( 125 I -Tyr-MIF-1) to bind to the solubilized proteins of isolated murine brain microvessels in the presence or absence of potential inhibitors. Sephadex chromatography separated bound from free labeled peptide. The binding was saturable as shown by inhibition with increasing concentrations of unlabeled Tyr-MIF-1. 125 I -Tyr-MIF-1 binding was not inhibited by an unrelated peptide or iodo-tyrosine. d-Tyr-MIF-1 had no effect, demonstrating the stereospecificity of the system. Met-enkephalin decreased the binding of 125 I -Tyr-MIF-1 to 84.4% of total, whereas Leu-enkephalin was without effect. Agonists for the mu, delta, and kappa opiate receptors did not change the binding, indicating that the proteins which bound to 125 I -Tyr-MIF-1 were not endogenous opiate receptors. The results indicate that, in vitro, Tyr-MIF-1 binds to brain microvessel proteins with characteristics similar to PTS-1.

Medicinal plants and Alzheimer's disease: Integrating ethnobotanical and contemporary scientific evidence.

The use of complementary medicines such as plant extracts in dementia therapy, varies according to the different cultural traditions. In orthodox Western medicine, contrasting with that in China and the Far East for example, pharmacological properties of traditional cognitive or memory enhancing plants have not been widely investigated in the context of current models of Alzheimer's disease. An exception is Ginkgo biloba in which the ginkgolides have antioxidant, neuroprotective, and cholinergic activities relevant to Alzheimer's disease mechanisms. The therapeutic efficacy of Ginkgo biloba extracts in Alzheimer's disease in placebo-controlled clinical trials is reportedly similar to currently prescribed drugs such as tacrine or donepezil and, importantly, undesirable side effects of Ginkgo biloba are minimal. Old European reference books (eg, medical herbals) document a variety of other plants such as Salvia officinalis (sage) and Melissa officinalis (balm) with memory improving properties, and cholinergic activities have recently been identified in extracts of these plants. Precedents for modern discovery of clinically relevant pharmacological activities in plants with long-established medicinal use include, for example, the interaction of alkaloid opioids in Papaver somniferum (Opium poppy) with endogenous opiate receptors in the brain. With recent major advances in understanding the neurobiology of Alzheimer's disease, and as yet limited efficacy of so-called rationally designed therapies, it may be timely to re-explore historical archives for new directions in drug development. This article considers not only the value of an integrative traditional and modern scientific approach to developing new treatments for dementia, but also in the understanding of disease mechanisms. Long before the current biologically based hypothesis of cholinergic derangement in Alzheimer's disease emerged, plants now known to contain cholinergic antagonists were recorded for their amnesic and dementia-inducing properties.

DNA Structure Determines Protein Binding and Transcriptional Efficiency of the Proenkephalin cAMP-responsive Enhancer

Two precisely arranged proenkephalin cAMP response elements (CREs) behave as a single protein binding site. The experiments described support a model in which a secondary structural change creates a new binding site, which is made up of sequences from both of the CREs. The CRE-binding protein (CREB) binds CRE-1, but binding there is entirely dependent on the presence of CRE-2. Electron spectroscopic images show that a CREB dimer occupies twice as much DNA in the proenkephalin gene as in the prodynorphin gene. The enhancer region is sensitive to P1 nuclease in a CREB concentration-dependent manner, and sensitivity is strand-specific, indicating protein-stabilized structural change. DNase I analysis shows that in the native proenkephalin gene, CREB binds both CRE-1 and CRE-2. In vivo, both CREs are occupied in the transcriptionally active proenkephalin gene, while neither is in the silent gene. Whereas CREB can bind CRE-2, mutation or elimination of either proenkephalin CRE alters response to second messengers and transcription factors. Thus, binding to CRE-2 alone is not sufficient. Specific and efficient transcription of the proenkephalin gene requires the presence of both CREs, precisely arranged to allow them to form a single protein binding site.

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to opioid receptors in frog brain membrane preparations.

A tritiated heptapeptide, [3H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([3H]Met-enkephalin-Arg6-Phe7), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent KD value of the radioligand calculated from homologous displacement experiments was 3.4 nM, and the maximal number of specific binding sites was 630 fmol/mg of protein. The KD determined from equilibrium saturation binding studies was found to be 3.6 nM. However, the Hill coefficient was far below unity (nH = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [3H]Met-enkephalin-Arg6-Phe7 binding reflects a kappa 2- and/or delta-subtype specificity of the heptapeptide. Binding to a kappa 1 and/or mu site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg6-Phe7 in frogs cannot be ruled out. The [3H]-Met-enkephalin-Arg6-Phe7 binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.

Central inhibition of nitric oxide synthase attenuates water intake but does not alter enhanced glucose utilization in the hypothalamo-neurohypophysial system of dehydrated rats

I.c.v. administration of a nitric oxide (NO) synthase inhibitor ( N G-monomethyl- l-arginine , NMMA, 500 μg/5 μl) to conscious rats deprived of water for 24 h attenuated drinking and decreased glucose utilization in the subfornical organ and median preoptic nucleus. NMMA did not alter the enhanced glucose utilization in the hypothalamo-neurohypophysial system (HNS) of dehydrated rats, although it has been shown to increase, selectively, oxytocin (OT) secretion [18]. This suggests that NO may act in the neural lobe to inhibit OT secretion and promote the preferential release of vasopressin during dehydration. This effect is similar to the blockade of endogenous opiate receptors by naloxone.

Activation of κ-opioid receptors in the nucleus ambiguus does not affect cardiovascular function or outcome following acute hemorrhage in the rat

The central cardiovascular response to hemorrhage is believed to be regulated, in part, by specific brain cardioregulatory nuclei, including the nucleus ambiguus (NA) of the hindbrain. Since endogenous opioid peptides and opiate receptors have been localized to this brain region, activation of endogenous opioid systems in the NA may affect the cardiovascular response to acute hemorrhage. The present study examined the effects of intracerebral microinjection of κ-receptor agonists into the NA prior to acute fixed-volume hemorrhage in awake rats. 15 min prior to fixed volume hemorrhage (7.5 ml/300 g), male Sprague-Dawley rats ( n = 59) received a microinjection of either (1) the synthetic κ-receptor agonist U-50, 488H (10 nM) or (2) U-50,488H (100 nM) or (3) the endogenous κ-receptor agonist dynorphin 1–17 (1 nM) or (4) Des-Tyr dynorphin 2–17, inactive at opiate receptors (1 nM) or (5) equal volume saline. With the exception of the first 10 min post-hemorrhage, where intracerebral injection of both dynorphin 1–17 and dynorphin 2–17 caused a transient suppression of mean arterial blood pressure ( P < 0.05 when compared to saline-treated controls), microinjection of the κ-agonists dynorphin 1–17 or U-50,488H had no effect on blood pressure, heart rate or mortality when compared to control animals. These results suggest that activation of κ-opiate receptors in the NA does not markedly influence cardiovascular response to acute hemorrhage.

Effects of an opiate receptor antagonist on renin release in dogs.

The present experiments were designed to determine whether blockade of endogenous opiate receptors with naloxone would suppress renin release induced by circulating epinephrine or by reductions of renal perfusion pressure. In the first series of experiments, anesthetized dogs were prepared with a flow probe around the left renal artery and a catheter in the left renal vein, permitting measurement of renin secretion before, during, and after 15-min infusions of epinephrine (50 ng.kg-1.min-1 iv). The epinephrine infusions were conducted either before or after blockade of opiate receptors with naloxone (1 mg/kg iv). Naloxone failed to alter the renin secretory response to intravenous epinephrine infusion. In a second series of experiments, anesthetized dogs were uninephrectomized and prepared with a constrictor cuff around the left renal artery and a renal arterial catheter distal to the cuff. After control measurements of renal perfusion pressure and plasma renin activity (PRA), the cuff was constricted at 15-min intervals to produce controlled stepwise reductions of renal perfusion pressure ranging from 15 to 90 mmHg. One-half of the animals was pretreated with naloxone (1 mg/kg iv). Naloxone pretreatment had no effect on the PRA response to reduced renal perfusion pressure at any pressure. The data fail to support the hypothesis that endogenous opioid peptides are modulators in the control of renin release.

Plasma catecholamine response to fetal hypoxemia is not potentiated by naloxone.

The interaction between the plasma catecholamine response to hypoxemia and endogenous opiate receptor blockade was investigated in 9 fetal lambs. The animals were randomly assigned to receive either normal saline (control group 1) or two doses of naloxone (group 2 = 0.5 mg/kg; group 3 = 1.0 mg/kg) by bolus injection followed by continuous infusion. Arterial PO2 decreased from 21 to 12 Torr and remained less than 14 Torr for 1 h. Arterial pH declined from 7.40 to a nadir of 7.32 at 1 h. Mean plasma catecholamine concentrations rose promptly from a baseline of 524-546 pg/ml for norepinephrine (NE) and 156-235 pg/ml for epinephrine (E) to reach a peak at 15 min (NE = 2,476-3,276, p less than 0.01; E = 1,856-4,065 pg/ml, p less than 0.01) and remained elevated thereafter. Neither dose of naloxone significantly altered plasma NE or E concentrations. Therefore, we conclude that endogenous opiates do not modulate the sympathoadrenal response to moderately long periods of hypoxemia unaccompanied by acidemia.

On the possible role of different endogenous opiate receptors in gastroduodenal ulceration of the rat

In Wistar rats of both sexes stress(restraint)- and drug (indomethacin)-induced gastric, as well, as cysteamine-induced duodenal ulceration was treated by morphine and nalorphine, given either alone or in combination. Neither morphine nor nalorphine had a significant anti-ulcerogenic effect. In contrast, the combined morphine + nalorphine treatment showed a highly significant anti-ulcerogenic action in the case of gastric ulceration but it was ineffective on duodenal ulcer formation. It seems that the chi and sigma endogenous opiate receptors play a significant role in the anti-ulcerogenic effect, while the mu receptors are involved to a considerably lesser degree. Moreover, the experimental results strengthen the view that gastric and duodenal ulceration are two different pathological entities and that ulcer formation is only a common consequence.

Unexpected Inhibitory Action ofN-Methyl-D,L-Aspartate on Luteinizing Hormone Release in Adult Ovariectomized Rhesus Monkeys: A Role of the Hypothalamic-Adrenal Axis*

N-methyl-D,L,-aspartate (NMA), an analog of the excitatory neurotransmitter aspartate, has been previously shown to acutely stimulate LH release in the rodent and primate. In this study, we examine the effect of NMA on LH secretion in the long term ovariectomized adult rhesus monkey. After a 3-h control period, three successive iv bolus injections of NMA (10 or 45 mg) were administered at hourly intervals, and LH and cortisol responses were compared with those after iv administration of physiological saline. LH concentrations remained unchanged throughout the saline infusion (n = 6) and during the 10-mg NMA injection regimen (n = 5). Unexpectedly, LH decreased during NMA injections at a dose of 45 mg (n = 10): areas under the LH curve, expressed as percentage of baseline control, were: hour 1, -16.0% (+/- 2.7 SE); hour 2, -28.4 (+/- 3.2 SE); hour 3; -30.9 (+/- 3.2 SE), P less than 0.005 vs. saline or 10 mg NMA. This inhibitory effect of NMA was prevented by the coadministration of GnRH (3 micrograms) (n = 5), suggesting that NMA acts at a suprapituitary level. Cortisol secretion was significantly increased by 45 mg of NMA; Total areas under the cortisol curve, expressed as percentage of baseline control, were: saline, -24.2% (+/- 4.2 SE); NMA (10 mg), -24.2 (+/- 2.0); NMA (45 mg), +22.2 (+/- 6.2); P less than 0.001 vs. NMA (10 mg) and saline, suggesting that NMA at the higher dose may activate the adrenal axis. To examine a possible role of the adrenal axis on NMA-induced LH inhibition, we next examined the effects of intraventricular administration of antiserum to CRF. Pretreatment with CRF antiserum prevented the decrease in LH levels seen during NMA (45 mg) in 4 of 8 monkeys (hour 2, -8.5% (+/- 6.5); hour 3, -10.3% (+/- 4.3); P less than 0.01 vs. NMA). The NMA-induced cortisol increase was prevented in the antiserum responsive but not in the nonresponsive animals. A similar preventive action on LH was seen after administration of the endogenous opiate receptor antagonist naloxone (2 or 5 mg/h), most notably in caged animals (n = 4: hour 1, 6.2% (+/- 3.8); hour 2, -2.8 (+/- 4.0); hour 3, -9.9 (+/- 5.0); P less than 0.005 vs. NMA, 45 mg, for hour 1 and hour 2). We conclude that the unexpected inhibitory effects of NMA on LH secretion in the adult ovariectomized monkey are the result of the activation of the hypothalamic-pituitary-adrenal axis by NMA and specifically of the release of CRF and endogenous opioid peptides.

Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study

An in vitro binding assay, using 125I-YLFQPQRFamide, a newly synthetized iodinated analog of FLFQPQRFamide, in which Phe 1 (F) has been substituted by a Tyr (Y), was developed to demonstrate and characterize putative binding sites of this brain morphine modulating peptide. This radioligand bound in a time-dependent manner to rat spinal cord membrane preparation. This binding was dose-dependent, saturable and reversible. Both kinetic data and saturation measured at equilibrium lead to the existence of a homogenous population of high affinity binding sites with a K d value of 0.09–0.1 nM and a maximal capacity B max of 14.5 ± 2fmol/mg protein. Results of competition experiments show that both FLFQPQRFamide and its analog YLFQPQRFamide had a similar capacity to inhibit the 125I-YLFQPQRFamide binding, suggesting that this radioiodinated analog is a good tool to study binding characteristics of FLFQPQRFamide receptors. The related octadecapeptide AGEGLSSPFWSLAAPQRFamide, another mammalian morphine modulating peptide competes for radioligand binding with similar potency. Our results also show that μ, δ and κ opiate receptor agonists as well as the antagonist naloxone were not able to affect binding either in presence or in absence of 120 mM NaCl. Together, these data demonstrate that FLFQPQRFamide does not function as an endogenous opiate receptor antagonist and that its capacity to reduce opiate-induced analgesia is supported by specific binding sites.

Effect of specific acute stressors on luteinizing hormone release in ovariectomized and ovariectomized estrogen-treated female rats.

In order to ascertain the influence of gonadal steroid hormones on the secretory response of the hypothalamic-hypophyseal luteinizing hormone (LH) axis to acute stress, the effects of four specific stressors on LH release were compared in ovariectomized versus ovariectomized steroid-treated rats. Groups of adult female Copenhagen-Fischer 344 rats were ovariectomized for either 1 or 2 weeks and exposed for specific intervals to one of the following stressors: novel environment, strobe light, restraint, or swim. Additional groups of animals were ovariectomized for 2 weeks and injected with 10 micrograms estradiol benzoate 24 and 48 h prior to exposure to the same stress stimuli. Multiple blood samples were obtained from these and nonstressed experimental controls at specific time points before, during, and after stress exposure. Transfer of 1-week ovariectomized rats to a novel environment, followed by a return to their original quarters 30 min later, resulted in a well-defined pattern of increased LH release. Novel environment stress also stimulated LH release in 2-week ovariectomized rats, as indicated by the comparison of mean LH values from the pre-stress versus post-stress sampling periods by paired test. Strobe light stress, on the other hand, had no effect on circulating LH in 1-week ovariectomized rats, but significantly increased mean post-stress plasma LH levels compared to mean pre-stress values in 2-week ovariectomized rats. While exposure to either 15 min of restraint or 10 min of swim stress had no effect on LH in rats ovariectomized for 1 week, both of these stressors resulted in a marked decline in LH release in 2-week ovariectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in micturition volume thresholds in conscious dogs following spinal opiate administration

Micturition difficulties appear as an often-reported side effect of the clinical use of opiates for spinal analgesia. Only a few experimental studies have focused specifically on this problem, especially in an unanesthetized animal model where chronic pharmacological studies can be carried out. The changes in micturition volume thresholds that occurred following spinal intrathecal injections of 1 mg of morphine sulphate were measured in 11 conscious dogs and compared with threshold changes produced in these same dogs by i.v. injections of various doses of morphine sulphate and by intrathecal and i.v. injections of naloxone HCl. In all cases, intrathecal or systemic morphine at doses of 1.0 mg or greater significantly ( P < 0.05) increased the bladder volume at which micturition took place. Naloxone, injected intrathecally to reverse the effects of intrathecal morphine, significantly reduced the micturition volume threshold, in most cases to below control volumes. A 400 μg dose of naloxone, injected intrathecally without prior injection of morphine, significantly lowered the volume threshold in 9 dogs, even though two of these dogs had elevated thresholds following naloxone injection. The reduction in volume thresholds by i.v. naloxone not preceded by morphine injection was not statistically significant over that of control. These results are interpreted in light of recent findings concerning localization of endogenous opiate receptors within the micturition reflex pathway.

An opiate component in realization of the vascular effects of clonidine

Systemic and regional hemodynamic changes (microsphere technique) were studied in urethan-chloralose anesthetized cats after intravenous clonidine and naloxone administration in different sequence. It was shown that antihypertensive effect of clonidine was caused by a decrease in the cardiac output and an increase in the total peripheral vascular conduction. The ability of clonidine to increase total peripheral conduction was completely blocked with naloxone preadministration. It was concluded that endogenous opiate receptors take part in the vasodilating clonidine effect in abdominal organs.

Effects of naloxone on basal and stimulated insulin secretion in the mouse

Endogenous opiates have been suggested to have effects on insulin secretion. In order to investigate the in vivo importance of endogenous opiate receptor agonists on basal and stimulated insulin secretion, the effect on plasma insulin of opiate receptor blockade by naloxone was studied in the mouse. It was found that the plasma insulin levels in naloxone-injected mice were moderately lower than in the control animals after 5 min. Ten minutes later, however, naloxone-injected animals had slightly higher plasma insulin levels than did the controls. Naloxone moderately potentiated the insulin secretory responses to glucose and the β 2-adrenoceptor agonist terbutaline. On the contrary, naloxone had no apparent effect on the insulin secretory response to the cholinergic agonist carbachol or the synthetic C-terminal octapeptide of cholecystokinin, CCK-8. In conclusion, endogenous opioid substances might moderate the regulation of basal insulin levels in the mouse. Further, they seem to have the capability to modulate the insulin release stimulated by glucose or β-adrenoceptor agonists.

Site-specific naloxone blockade of brain self-stimulation duration

Moderate doses of naloxone (1, 5, 10 mg/kg) did not reduce response rate measured by the initiation of intracranial self-stimulation bursts to the medial forebrain bundle or central gray, at several current intensities (30, 60, or 90 μA). Raising current intensity increased rates and duration of lever pressing at both sites. Naloxone produced a dose dependent reduction in self-selected current duration at both sites averaged over intensity. However, naloxone completely blocked the effect of intensity only at the central gray site. In contrast to research showing effects of only high naloxone doses (e.g., 40 mg/kg) on self-stimulation at opiate-rich and opiate-poor receptor sites, this study indicates that endogenous opiates and opiate receptor mechanisms interact with aversive properties to modify central gray self-stimulation.