Spinal cord-mediated opiate effects on gastrointestinal transit in mice.
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Animals↗
Biomedical subjects
F Porreca
Publications and source records attributed to F Porreca.
A comparison of the receptor constants of morphine and ethylketocyclazocine for analgesia and inhibition of gastrointestinal transit in the rat.
The efficacies and dissociation constants of proposed mu and kappa receptor agonists (morphine and ethylketocyclazocine, respectively) were compared using the method of partial irreversible blockade (with buprenorphine) and Stephenson's theory of drug action. While there was good agreement between the dissociation constant (KA) of morphine in analgesia (3.3 x 10(-5) M) and in inhibition of gastrointestinal transit (1.1 x 10(-5) M), the KA of ethylketocyclazocine differed by an order of magnitude in these endpoints (3.2 x 10(-6) M and 6.7 x 10(-5) M, respectively). The efficacies of morphine were found to be similar for the two effects studied (4.23 and 5.26), while those for ethylketocyclazocine differed markedly (2.06 and 10.39). The fraction of receptors remaining unblocked after buprenorphine was consistent for the test but not for the agonist, indicating a different distribution of receptors for the two endpoints. Our results strongly suggest that morphine induces analgesia, and slows transit in the small intestine, through the same type of receptor. The same conclusion cannot be drawn for ethylketocyclazocine.
Analgesia↗
Morphine-receptor dissociation constant and the stimulus-effect relation for inhibition of gastrointestinal transit in the rat.
The dissociation constant (KA) of morphine for its receptors was determined by the method of partial irreversible blockade of the receptor population using inhibition of gastrointestinal transit of a forced charcoal meal as the pharmacological endpoint. The anti-motility effect of morphine was antagonized when rats were pretreated with buprenorphine (0.3 mg/kg s.c.), a narcotic antagonist analgesic, 30 min before morphine and the extent of gastrointestinal transit was estimated a further 45 min later. With this schedule of drug administration, the agonist action of buprenorphine is minimal and its antagonist action predominates. The value of KA was (1.1 +/- 0.2) x 10(-5) mol/kg, a value close to that previously reported (2.9 x 10(-5) mol/kg) by us with these compounds in the rat tail flick test. The value of [A50], found here was 2.15 x 10(-6) mol/kg, approximately 1/5 of that of KA. Also, the stimulus-effect relation of the tissue, defined in Stephenson's theory, was plotted and found to be nonlinear. This result, when coupled with the inequality of KA and [A50], argues against the application of classical drug-receptor theory to this system. The apparent agreement between KA values for antinociception and inhibition of gastrointestinal transit is interesting, but does not necessarily prove equivalent receptors mediating the two different effects.
Animals↗
Differentiation of apomorphine from bromocriptine, piribidel and TRH by chronic administration in rats.
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Agonistic Behavior↗
Splenic capping: an experimental study of a new technique for splenorrhaphy using woven polyglycolic acid mesh.
The use of polyglycolic acid (PGA) stretchable mesh applied to the experimentally injured canine spleen can achieve satisfactory immediate hemostasis by tamponade and simplifies the use of sutures to control remaining areas of hemorrhage. PGA mesh with 1/4" and 1/8" openings was utilized for splenorrhaphy in 12 adult mongrel dogs subjected to sharp splenic trauma. By gross and histologic examination, the PGA mesh material appears to undergo progressive absorption to complete absorption by 85 days. For the 12 animals and 30 operative procedures the only complications of the use of the mesh were the occurrence of an intrasplenic seroma in a single animal at 6 weeks after operation and three wound infections. With this material, rapid, simple canine splenic injury repair can be achieved. PGA mesh further assists in the healing process, and in maintaining maximum splenic architecture and function. The material used in this study was manufactured and supplied by Davis & Geck, American Cyanamid, Danbury, Connecticut.
Animals↗
Tolerance and cross-tolerance studies with morphine and ethylketocyclazocine.
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Animals↗
On the criteria for classifying opiate agonists in rats.
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Analgesics↗
Time course of antagonism of morphine antinociception by intracerebroventricularly administered naloxone in the rat.
The kinetic profile and half-life of naloxone were studied for possible use in determination of pA2 and KB in vivo. Rats were given morphine subcutaneously and after 15 min naloxone or saline, intracerebroventricularly. A further 15 min later, and at 15 min intervals up to 135 min after morphine, the animals were tested for analgesia in the tail flick test. The dose-response curves of the naloxone group were shifted to the right of those for the saline group. The amount of displacement decreased with time, indicative of the disappearance of naloxone. The graph of log (dose ratio-1) vs. time was linear with negative slope, in agreement with the time-dependent form of the equation for competitive antagonism. From this slope, the half-life of naloxone was calculated to be 13.3 min. These results demonstrate that the time-dependent method is useful in obtaining the kinetics of centrally acting opiate antagonists.
Analgesia↗
Extraction and measurement of morphine: correlation of brain level and s.c. dose in drug-naive and morphine-tolerant rats.
A method was recently developed by Raffa et al (1) for rapid analysis of brain levels of morphine in rats given the drug subcutaneously. The technique combines the extraction procedure of Sprague and Takemori (2) and the HPLC methodology of Peterson et al (3). The purpose of the present work was verification of the accuracy of this technique and its application to an accompanying study in which the dissociation constants of morphine determined in drug-naive and morphine-tolerant rats were compared. Male, Sprague-Dawley rats (180-220 g) were given morphine sulfate s.c. 60 min prior to testing. Each rat in the "tolerant" group received two 75 mg morphine pellets subcutaneously which were removed 96 h later. Brain levels of morphine were measured a further 24 h later. Morphine levels in rat brain ranged from 52 to 1800 ng, corresponding to subcutaneous doses of 2.5 to 320 mg/kg. This range of brain levels agrees well with those obtained using different methods (4,5). We found no significant difference in brain levels of morphine in naive and tolerant rats given the same doses of morphine (10, 20, 40, and 80 mg/kg, s.c.). Thus, in the determination of dissociation constants for morphine, the same relation can be used for both naive and morphine-tolerant rats when converting administered dose to brain level.
Animals↗
Estimation in vivo of the receptor constants of morphine in naive and morphine-tolerant rats.
The efficacy and dissociation constant of morphine in naive and morphine-tolerant rats were estimated by the method of partial irreversible blockade of a fraction of the receptor population with buprenorphine. The dissociation constant (KA) of morphine increased from 3.3 x 10(-5) M in naive to 1.4 x 10(-4) M in morphine-tolerant animals, indicating a decrease in the affinity of morphine for its receptor in the tolerant state. The efficacy of morphine (KA/A50 + 1) was constant in naive and tolerant animals (4.23 and 4.46, respectively). When the data were recalculated following conversion of administered dose to brain morphine concentration, the value of KA was 1.7 x 10(-7) M in naive and 7.7 x 10(-7) M in morphine-tolerant rats, while the efficacy was 2.5 and 3.4, respectively. In addition, the stimulus-effect relationship varied in the two states, with the curve in the tolerant animal being of different shape and broader range than in the naive rat. The present results suggest that (a) tolerance to opiate agonists may involve affinity changes and (b) post-receptor events leading to the measured effect may also be affected.
Analgesia↗
Estimation of the dissociation constant of naloxone in the naive and morphine-tolerant guinea-pig isolated ileum: analysis by the constrained Schild plot.
The pA2 values for naloxone-morphine were determined using ilea taken from naive and morphine-tolerant guinea-pigs. This constant decreased from 8.56 (+/- 0.22) in the naive to 7.95 (+/- 0.12) in tolerant tissues. The values of the pA2's were based on Schild plots using conventional regression techniques; this regression resulted in values of -log KB of 8.79 (+/- 1.5) and 8.18 (+/- 1.2) in naive and morphine-tolerant ilea, respectively. While pA2 differed, the difference in -log KB was not statistically significant. Since these two values are theoretically equivalent, the above finding appears paradoxical. Constraining the slope of the Schild plot to the theoretically required -1 resolved this discrepancy by reducing the error inherent in the calculation of -log KB determined by conventional regression.
Animals↗
Effects of a novel opioid peptide antagonist on rat bladder motility in vivo.
The agonist, and opioid antagonist, effects of intracerebroventricularly (ICV) given D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), a cyclic analogue of somatostatin octapeptide, were evaluated using the micturition reflex of the anesthetized rat as the endpoint. Antagonist effects were evaluated against equieffective doses of selective mu [D-Ala2,NMPhe4,Gly-ol]enkephalin (DAGO) and delta [D-Pen2,D-Pen5] enkephalin (DPDPE) opioid agonists. At low ICV doses, CTP preferentially antagonized DPDPE rather than DAGO; increasing the dose of CTP further effectively antagonized both mu and delta agonists, while even higher doses showed an agonist effect alone which was not blocked by adrenergic, cholinergic or opioid antagonists. Selective opioid antagonist doses of CTP failed to block the inhibition of the micturition reflex produced by pentobarbital. Possible residual somatostatin like properties of CTP were tested by using somatostatin as a possible antagonist of equieffective doses of DPDPE and DAGO; somatostatin did not antagonize these agonists. Repeated exposure to CTP resulted in the development of acute tolerance to the agonist effect, and also prevented the inhibition of the reflex by high doses of somatostatin, with the converse experiment showing a similar pattern; thus, repeated somatostatin resulted in tolerance and subsequent cross-tolerance to the agonist effects of CTP. In animals tolerant to somatostatin, CTP nevertheless behaved as an opioid antagonist. The present results indicate that CTP possesses opioid antagonist properties in vivo which are pharmacological in nature but nevertheless retains residual somatostatin-like activity at higher doses.
Animals↗
Peptide fragments derived from the beta-chain of hemoglobin (hemorphins) are centrally active in vivo.
A novel tetrapeptide (hemorphin-4) and pentapeptide (hemorphin-5), derived from the beta-chain of hemoglobin, were synthesized by solid-phase methodology, purified and the amino acid sequences confirmed. The central (ICV) effects of hemorphin-4 and -5 were studied in two models of phasic and tonic nociception, the mouse warm water tail-flick assay and hindpaw formalin assay, respectively. Additionally, two physiological endpoints, central modulation of bladder motility and central effects on intestinal propulsion, were studied in rats and mice, respectively. In the tail-flick assay, both peptides (40-100 nmoles) produced a dose-related naloxone-reversible antinociceptive effect when tested 10 min after peptide administration, with the tetrapeptide being slightly more potent than the pentapeptide. No effect was noted for either peptide using the tonic nociception assay, except at a dose of 150 nmoles for hemorphin-5. Inhibition of gastrointestinal propulsion was also not affected by either peptide. However, both peptides (10-40 nmoles) inhibited micturition contractions in a dose-related and naloxone-reversible fashion, with the tetrapeptide being twice as potent as the pentapeptide. These findings provide evidence that hemorphin-4 and -5 exert naloxone-reversible opioid actions in vivo and, therefore, may be physiologically important blood-borne peptides.
Analgesics↗
Peptide targeting and delivery across the blood-brain barrier utilizing synthetic triglyceride esters: design, synthesis, and bioactivity.
As an approach to the development of therapeutically useful peptide pharmaceuticals that can penetrate the blood-brain barrier, we have designed and demonstrated the application of a carrier-targeting system. We have developed a prodrug design strategy that is designed to utilize membrane-bound enzymes whereby release of a bioactive peptide from a highly lipophilic triglyceride peptide-carrier is achieved in situ, thus attaining high localized concentrations of the bioactive peptide. Following localization of such a system, normal peptidase and lipase action is utilized to release the active peptide (deltorphin II) intact and in high concentration. At present, the exact mechanisms are unclear, but the observed results in which analgesia is observed following peripheral administration suggest that the active peptide is able to cross the blood-brain barrier and sustain prolonged periods of analgesia as determined by antinociception tests by release of the bioactive peptide. In vitro tests of binding and bioactivity by the peptide conjugate show essentially no potency in either target or control analogues, but potent antinociceptive effects are observed following peripheral administration.
Analgesia↗
Treatment with antisense oligodeoxynucleotide to a conserved sequence of opioid receptors inhibits antinociceptive effects of delta subtype selective ligands.
Previous work has suggested the existence of subtypes of the delta opioid receptor (DOR) which have been termed delta1 and delta2. [D-Ala2, Glu4]deltorphin has been suggested to selectively elicit antinociception via the delta2 receptor while [D-Pen2, D-Pen5]enkephalin (DPDPE) is thought to act via the delta1 receptor. Treatment with an antisense oligodeoxynucleotide (oligo) directed towards the N-terminal portion of the cloned DOR has been demonstrated to selectively inhibit the antinociceptive actions of [D-Ala2, Glu4]deltorphin, but not of DPDPE, suggesting that the cloned DOR corresponds to that pharmacologically defined as delta2. Here, an antisense oligo (or a mismatch sequence) was designed to target a conserved region of the cloned mu, delta and kappa opioid receptor. These oligos were employed in order to determine whether the antinociceptive effects of [D-Ala2, Glu4]deltorphin, as well as DPDPE, could be inhibited. The data indicate that the antinociceptive actions of both ligands were inhibited by treatment with this antisense, but not with the mismatch oligo. Taken together, the results of the treatments with oligos directed towards the N-terminal portion of the cloned DOR and with that directed to the conserved region of the opioid receptors suggest that (a) DPDPE effects are mediated by a subtype of the DOR which shares a domain common to the cloned opioid receptors, and (b) the N-terminal region differs between these putative DOR subtypes.
Animals↗