Enhanced antinociceptive response to intracerebroventricular kyotorphin in Pept2 null mice.

L-Kyotorphin (L-KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton-coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain of L-KTP and its synthetic diastereomer D-kyotorphin (D-KTP) in wild-type and Pept2 null mice. CSF clearance of L-KTP was slower in Pept2 null mice than in wild-type animals, and this difference was reflected in greater L-KTP-induced analgesia in Pept2 null mice. Moreover, dose-response analyses showed that the ED50 of L-KTP in Pept2-deficient animals was one-fifth of the value observed in Pept2-competent animals (4 vs. 21 nmol for null vs. wild-type mice, respectively). In contrast, the ED50 of D-KTP was very similar between the two genotypes (9-10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects of L-KTP and D-KTP. The enhanced antinociceptive response to L-KTP in Pept2 null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates.

Big dynorphin as a putative endogenous ligand for the kappa-opioid receptor.

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.

Research on the neurophysiological mechanisms of acupuncture: review of selected studies and methodological issues.

This presentation reviews studies that contribute to an understanding of the neurophysiological mechanisms of acupuncture. A 1973 study, using volunteer medical students, looked into acupuncture's analgesic effect on experimentally induced pain and suggests that humoral factors may mediate acupuncture-induced analgesia. In a study of the possible role of the cerebrospinal fluid transmission of pain suppression effects of acupuncture, cerebrospinal fluid from acupuncture-treated rabbits was infused into recipient rabbits. The analgesic effect was observed in the recipient rabbits, suggesting that acupuncture-induced analgesia may be mediated by substances released in the cerebrospinal fluid. Studies of electroacupuncture in rats revealed that both low-frequency and high-frequency stimulation could induce analgesia, but that there are differential effects of low- and high-frequency acupuncture on the types of endorphins released. In another study, low-frequency electroacupuncture, given as median nerve stimulation in cats, was shown to protect the myocardium by inhibiting sympathetic pressor response and increasing myocardial oxygen demand. The development of neuroimaging tools, such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), make noninvasive studies of acupuncture's effects on human brain activity possible. Studies using PET have shown that thalamic asymmetry present among patients suffering from chronic pain was reduced after the patients underwent acupuncture treatment. Other studies, using fMRI, have pointed to relationships between particular acupoints and visual-cortex activation. These powerful new tools open the possibility to new scientific studies of this ancient therapy.

Cognitive functioning and cerebrospinal fluid concentrations of neuropeptides for patients with good neurological outcomes after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: Many patients exhibit cognitive disturbances after aneurysmal subarachnoid hemorrhage (SAH). Structural and functional neuroimaging has failed to demonstrate any correlation with these complaints. This study was performed to investigate whether neuropeptide concentrations in cerebrospinal fluid could be related to cognitive disturbances after SAH. METHODS: Lumbar cerebrospinal fluid was obtained, 3 to 6 months after surgery, from 17 patients who experienced good outcomes after aneurysmal SAH. The samples were analyzed for various neuropeptides using radioimmunoassays, and the peptide concentrations were evaluated in relation to scores on standardized neuropsychological tests. RESULTS: The neuropsychological test results were normal for eight individuals, whereas the remaining nine patients exhibited various degrees of cognitive impairment. There was no correlation between the concentrations of arginine vasopressin or neuropeptide Y and test performance. However, significant correlations between cognitive impairment and elevated levels of beta-endorphins (P = 0.02), corticotropin-releasing factor (P = 0.004), and delta sleep-inducing peptide (P = 0.045) were noted. CONCLUSION: Patients with cognitive impairments after aneurysmal SAH exhibited higher cerebrospinal fluid concentrations of endorphins, corticotropin-releasing factor, and delta sleep-inducing peptide than did those with normal capacity. This is probably attributable to diffuse derangement of transmitter release in the brain, resulting from the insult or ensuing complications, although a secondary increase in corticotropin-releasing factor concentrations caused by increased stress during the testing because of reduced cognitive capacity cannot be excluded.

Electroacupuncture: mechanisms and clinical application.

Acupuncture is an ancient Chinese method to treat diseases and relieve pain. We have conducted a series of studies to examine the mechanisms of this ancient method for pain relief. This article reviews some of our major findings. Our studies showed that acupuncture produces analgesic effect and that electroacupuncture (EA) is more effective than manual acupuncture. Furthermore, electrical stimulation via skin patch electrodes is as effective as EA. The induction and recovering profiles of acupuncture analgesia suggest the involvement of humoral factors. This notion was supported by cross-perfusion experiments in which acupuncture-induced analgesic effect was transferred from the donor rabbit to the recipient rabbit when the cerebrospinal fluid (CSF) was transferred. The prevention of EA-induced analgesia by naloxone and by antiserum against endorphins suggests that endorphins are involved. More recent work demonstrated the release of endorphins into CSF following EA. In addition, low frequency (2 Hz) and high frequency (100 Hz) of EA selectively induces the release of enkephalins and dynorphins in both experimental animals and humans. Clinical studies suggesting its effectiveness for the treatment of various types of pain, depression, anxiety, spinally induced muscle spasm, stroke, gastrointestinal disorders, and drug addiction were also discussed.

cGMP and cAMP in prostaglandin-induced pial artery dilation and increased CSF opioid concentration.

It has been observed that prostaglandins (PG) PGE2 and PGI2 increased cortical periarachnoid cerebrospinal fluid (CSF) methionine enkephalin (Met-enk) and leucine enkephalin (Leu-enk) concentrations in the newborn pig. It was also observed that PG-induced pial artery dilation was associated with elevated CSF guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) levels in the piglet. However, other studies have not always supported a role for cGMP in PG dilation. The present study used a pharmacological approach to test the hypothesis that both cGMP and cAMP contribute to PG-induced pial dilation and associated elevated CSF opioid concentration. PGE2 produced pial vasodilation that was blunted by the Rp diastereomer of bromoguanosine 3',5'-cyclic monophosphothioate [Rp-8-BrcGMPS (10(-5)M)], a cGMP antagonist (9 +/- 1, 16 +/- 1, and 23 +/- 1 vs. 4 +/- 1, 6 +/- 1, and 9 +/- 1% for 1, 10, and 100 ng/ml PGE2 before and after Rp-8-BrcGMPS, respectively). PGE2 elevated CSF Met-enk concentration, and these biochemical changes were also blunted by Rp-8-BrcGMPS (1,001 +/- 23, 1,424 +/- 54, and 1,973 +/- 56 vs. 804 +/- 41, 988 +/- 52, and 1,222 +/- 21 pg/ml for control, 10, and 100 ng/ml PGE2 in the absence and presence of Rp-8-BrcGMPS, respectively). Similar biochemical and vascular effects of Rp-8-BrcGMPS were observed for PGI2. Additionally, the Rp diastereomer of bromoadenosine 3',5'-cyclic monophosphothioate [Rp-8-BrcAMPS (10(-5)M)], a cAMP antagonist, blunted PGE2 dilation (10 +/- 1, 15 +/- 1, and 24 +/- 1 vs. 5 +/- 1, 8 +/- 1, and 12 +/- 1% for 1, 10, and 100 ng/ml PGE2 before and after Rp-8-BrcAMPS, respectively). PGE2-associated increases in CSF Met-enk and Leu-enk were similarly blunted by Rp-8-BrcAMPS. These data show that both cGMP and cAMP contribute to PG-induced pial dilation and that PG-associated elevated CSF cGMP and cAMP levels result in increased CSF Met-enk and Leu-enk concentration.

Endogenous opioids and opiate antagonists in autism: brief review of empirical findings and implications for clinicians.

Endogenous opioid dysfunction hypotheses for the development of autism are reviewed, along with clinical empirical studies of opiate antagonists in autism and self-injurious behaviour. There is not yet sufficient evidence to suggest the use of opiate antagonists in the treatment of autism. Further research, particularly of natrexone in severe self-injury, is warranted.

The role of opioids in newborn pig fluid percussion brain injury.

The present study was designed to characterize the relationship between cerebral opioid concentration, cerebral hemodynamics, and cerebral oxygenation following percussion brain injury in neonatal pigs. Previous research found that opioids represent a significant vasoactive component in the regulation of the neonatal piglet cerebral circulation. Anesthetized newborn (1-5 days old) pigs equipped with a closed cranial window were connected to a percussion device consisting of a saline-filled cylindrical reservoir with a metal pendulum. Brain injury of moderate severity (1.9-2.3 atm.) was produced by allowing the pendulum to strike a piston on the cylinder. Fluid percussion brain injury decreased pial arteriolar diameter (132 +/- 5 to 110 +/- 5 microns within 10 min). Cerebral blood flow also fell within 10 min of injury and continued to fall progressively for 3 h, resulting in a 46 +/- 4% decrease. Within 30 s of brain injury, there was a transient increase in cerebral hemoglobin-O2 saturation that was reversed to a progressive profound decrease in cerebral hemoglobin-O2 saturation for the next 3 h, as measured by near infrared spectroscopy. CSF opioid concentrations were increased 10 min after brain injury; dynorphin showed the largest proportional increase (5.8 +/- 0.9 fold). The CSF concentration for other opioids continued to increase over 180 min while the dynorphin concentration progressively decreased with time. In naloxone (1 mg/kg i.v.) pretreated piglets, the brain injury induced decrease in arteriolar diameter was attenuated (129 +/- 5 to 121 +/- 5 microns within 10 min). Similarly, the decrease in regional cerebral blood flow and cerebral hemoglobin-O2 saturation observed following brain injury were also blunted by naloxone.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the levels of several endogenous opioid peptides in dog cerebrospinal fluid following morphine administration.

The cisterna magna of dogs anesthetized with sodium Surital was fitted with a cannula, and cerebrospinal fluid (CSF) was withdrawn before (control) and one hour after the s.c. injection of 10 mg/kg of morphine sulfate (morphine). The CSF from control and morphine-treated dogs was purified initially by gel filtration. Each fraction was submitted to opiate bioassay procedures, followed by high performance liquid chromatography (HPLC) purification on a mu-Bondapak C18 column. Two of the CSF fractions from HPLC purification showed greater opiate-like activity after morphine treatment than that in controls. One fraction contained morphine, the other an unknown peptide. This latter fraction produced a dose-dependent effect in the mouse tail-flick test. This fraction did not show radioimmunoreactivity to methionine (met)- or leucine (leu)-enkephalins, but showed a small amount of reactivity to beta-endorphin and dynorphin (1-13). Further purification of this fraction by HPLC yielded a fraction with five peaks, which upon amino acid analysis were found to contain small peptides. Met- and Leu-enkephalins, beta-endorphin and dynorphin (1-13)-like immunoreactivity in the fraction in which the respective standard was eluted by HPLC was significantly increased after a single administration of morphine. Based on these results, it is suggested that morphine at an antinociceptive dose causes the release of endogenous opioid peptides and may also stimulate the biosynthesis of their precursor molecules, pre-pro-opiomelanocortin, pre-pro-enkephalin A and pre-pro-enkephalin B.

[Kyotorphin like substance in human cerebrospinal fluid of patients with persistent pain].

Kyotorphin is an analgesic neuropeptide isolated from the bovine brain in 1979. Further studies showed that kyotorphin produces an analgesia through an increased release of met-enkephalin in the brain and the spinal cord. We showed that it is also found in the human cerebrospinal fluid and the concentrations of kyotorphin in normal human CSF is 1.19 +/- 0.51 pmol.ml-1. We also found that it is lower in patients with persistent pain (0.24 +/- 0.04 pmol.ml-1). Above results suggest that kyotorphin acts as a putative neuromediator and/or an endogenous pain modulator in the human brain.

Levels of dynorphin peptides, substance P and CGRP in the spinal cord after subchronic administration of morphine in the rat.

Rats were rendered dependent on morphine by repeated injections of morphine, in increasing doses for 14 days and sacrificed. Levels of peptides in the dorsal spinal cord and dorsal root ganglia were analyzed in rats decapitated 2 hr, 24 hr (acute abstinent) or 7 days (late abstinent) respectively, after the last injection of drug. Dynorphin A was significantly decreased in rats abstinent for 24 hr, while dynorphin B remained unaffected. Substance P and CGRP, both putative transmitters in nociceptive primary afferent neurones, and partly existing together in the same neurone, were affected differently. Significantly less substance P but unchanged levels of CGRP were detected in rats abstinent for 24 hr, while on the other hand, CGRP but not levels of substance P, were increased 2 hr after the final injection. In dorsal root ganglia, levels of substance P were lower at 2 hr, while levels of CGRP were unaffected. In late (7 days) abstinence, no effect of opiate on any peptide was detected.

Opioids in cerebrospinal fluid in hypotensive newborn pigs.

This study was designed to determine if opioids were detectable in cerebrospinal fluid (CSF) and if these concentrations were altered by hemorrhagic hypotension. This study was further designed to determine the effects of topically administered opioids on pial arteriolar diameter during normotension and hypotension. Closed cranial windows were used to determine pial arteriolar diameter. Periarachnoid cortical and cisterna magna CSF was collected from piglets during normotension and hypotension (systemic arterial pressure decreased from 63 +/- 1 to 33 +/- 1 mm Hg). Opioid profiles were assessed qualitatively by radioreceptor assay, and individual opioids were measured quantitatively by radioimmunoassay. Periarachnoid cortical and cisterna magna CSF methionine enkephalin-, leucine enkephalin-, dynorphin-, and beta-endorphin-like receptor active values all were increased by hypotension. When quantified by radioimmunoassay, periarachnoid cortical CSF values for methionine enkephalin-like immunoreactivity were 1,167 +/- 58 and 2,975 +/- 139 pg/ml for normotension and hypotension, respectively. Periarachnoid cortical CSF radioimmunoassay values for dynorphin-like immunoreactivity were 15 +/- 2 and 28 +/- 2 pg/ml for normotension and hypotension, respectively. When applied topically to the cortical surface, synthetic methionine enkephalin increased pial arteriolar diameter (134 +/- 4, 158 +/- 4, and 163 +/- 4 microns for control, 574 pg/ml [10(-10) M], and 5,740 pg/ml [10(-9) M], respectively). Similarly, topical synthetic leucine enkephalin and dynorphin elicited pial arteriolar dilation. However, beta-endorphin produced arteriolar constriction. Hypotension attenuated methionine and leucine enkephalin-induced dilation and reversed dynorphin-induced dilation to concentration-dependent constriction. beta-Endorphin-induced constriction was not changed by hypotension. Therefore, opioids could contribute to the control of the cerebral circulation during hypotension.

Stereoselective effect of morphine on antinociception and endogenous opioid peptide levels in plasma but not cerebrospinal fluid of dogs.

Morphine releases endogenous opioids into the circulation of dogs. To test the stereospecificity of this effect, as well as to determine whether morphine also releases endogenous opioids centrally, which might be involved in its antinociceptive action, the effects of (-)-morphine sulfate (10 mg/kg, sc) or (+)-morphine hydrobromide on antinociception in a dog tail-flick test, on semi-quantified morphine-induced signs of salivation, emesis, defecation and ataxia, and on the plasma and cerebrospinal fluid (CSF) levels of endogenous opioid peptides were studied. Plasma and CSF levels of immunoreactive beta-endorphin (i-BE), met-enkephalin (i-ME), leu-enkephalin (i-LE), and dynorphin (i-DY) were quantified by radioimmunoassay in octadecylsilyl-silica cartridge extracts. Immunoreactive morphine (i-M) levels were measured in unextracted samples. (-)-Morphine treatment significantly increased antinociception, morphine-induced signs, i-M levels in plasma and CSF, and i-BE, i-ME, and i-LE levels in plasma, but not CSF. Levels of i-DY remained constant in plasma and CSF. (+)-Morphine treatment did not alter any of these parameters, indicating that the effects of morphine on nociception, behavioral signs, and plasma endogenous opioids in dogs were stereoselective. It is concluded that morphine does not cause an increase in immunoreactive endogenous opioid peptides in the CSF at the time of its peak antinociceptive effect.

N-terminal degradation of low molecular weight opioid peptides in human cerebrospinal fluid.

Opioid peptides are present in human cerebrospinal fluid (CSF), and their levels are reported to change in some pathologic conditions. However, less is known about their degradation in CSF. In the present study, human CSF was found to contain aminopeptidase activity which hydrolyzed alanyl-, leucyl- and arginyl-naphthylamides in a ratio of 100:28:27. Twelve CSF samples hydrolyzed alanyl-2-naphthylamide and degraded Met5-enkephalin (N-terminal hydrolysis) at rates of 188 +/- 38 and 420 +/- 79 pmol/min/mL respectively. Further, the distribution of alanyl-naphthylamidase activity in individual samples (39-437 pmol/min/mL) was closely correlated with that of Met5-enkephalin degradation (37-833 pmol/min/mL). Both alanyl-naphthylamidase and enkephalin degradation were optimal at pH 7.0 to 7.5 and were inhibited by aminopeptidase inhibitors amastatin (IC50 = 20 nM), bestatin (4-7 microM) and puromycin (30-35 microM). Conversely, degradation was unaffected by inhibitors of neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin converting enzyme (captopril). The Km of Met5-enkephalin for the CSF aminopeptidase activity was 201 +/- 19 microM (N = 4). Rates of hydrolysis of the Tyr1-Gly2 bond of larger opioid peptides decreased with increasing peptide length. Pooled, concentrated CSF hydrolyzed Leu5-enkephalin, dynorphin A fragments [1-7], [1-10] and [1-13] and dynorphin A at rates of 2.05 +/- 0.27, 1.27 +/- 0.18, 0.94 +/- 0.06, 0.55 +/- 0.14 and 0.16 +/- 0.03 nmol/min/mL respectively. When analyzed by rocket-immunoelectrophoresis against antisera to aminopeptidase M (EC 3.4.11.2), the concentrated CSF formed an immunoprecipitate which could be stained histochemically for alanyl-naphthylamidase activity. These data are consistent with a significant role for aminopeptidase M activity in the degradation of low molecular weight opioid peptides in human CSF.

Beta-endorphin in cerebrospinal fluid and serum after severe head injury.

Endogenous opioids have been implicated as a cause of secondary damage after neural injury. The basis for this statement is primarily indirect evidence from studies that demonstrate neurological or physiological improvement when opiate antagonists are given. This study directly evaluates the level of the endogenous opioid beta-endorphin in 15 patients with severe head injury. Levels of immunoreactive beta-endorphin (ir-beta E) from ventricular cerebrospinal fluid (vCSF) and serum were determined less than 24 hours after trauma. No significant correlation was found with the degree of initial injury, age, sex, or either 6- or 12-month outcome. Levels of ir-beta E from vCSF were significantly lower in patients who had received intravenous administration of morphine sulfate (48.4 +/- 5.8 versus 85.9 +/- 10.1 pg/ml, P = 0.008). No correlation was found between vCSF levels of ir-beta E and elapsed time after injury. Although vCSF and serum ir-beta E were correlated (r = 0.532, P = 0.050), the latter exhibited a different profile; the mean level of serum ir-beta E was not significantly different in those patients who received morphine, and serum ir-beta E had a significant negative correlation with time after injury (r = -0.587, P = 0.03). These results do not support a relationship between acute levels of vCSF or serum ir-beta E and the degree of neurological injury or outcome after severe head trauma. This article, therefore, is a contribution to the body of literature in which the purported detrimental effect of beta-endorphin was not demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-methyldopa induces a naltrexone-insensitive antinociception and hypomotility in rats.

1. This study served to investigate whether endogenous opioid peptides play a role in the putative antinociceptive and the sedative actions of alpha-methyldopa. 2. In conscious normotensive rats, alpha-methyldopa induced hypotension, starting around 1 h and reaching a maximum 3-4 h after administration. Pretreatment with naltrexone resulted in an inhibition of alpha-methyldopa-induced hypotension. 3. alpha-Methyldopa dose-dependently increased hot plate latency which became evident after a 4 h lag period and reaching a maximum effect at 6 h. The antinociceptive effect of alpha-methyldopa was not affected by naltrexone. 4. In a small open field, alpha-methyldopa dose-dependently suppressed locomotion and sniffing behaviour. These effects of alpha-methyldopa were apparent 1 h after administration and were naltrexone-insensitive. 5. No changes in the level of beta-endorphin-like immunoreactivity in plasma and cerebrospinal fluid were observed after administration of alpha-methyldopa. 6. The results indicate that endogenous opioid peptides are involved in the hypotensive action of alpha-methyldopa but not in alpha-methyldopa-induced hypomotility and antinociception.