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J M Musacchio

Publications and source records attributed to J M Musacchio.

At least 55 records · Page 3Linked to original sources
PubMed1984

The effect of tolerance on opiate dependence as measured by the adenylate cyclase rebound response to naloxone in the NG108-15 model system.

The naloxone-induced adenylate cyclase rebound response produced by opiates in the NG108-15 cells is quite apparent after 48 h treatment with M but it is undetectable after incubation with the high intrinsic activity agonist etorphine. Replacement of agonists by antagonists in receptors that have been rendered inactive by tolerance does not elicit the AC rebound response, which is considered a biochemical correlate of dependence. This indicates that marked tolerance and receptor down-regulation induced by prolonged treatment with high efficacy agonists may reduce the level of dependence.

Adenylyl Cyclases↗
PubMed1984

Specific binding of a 125I-labeled substance P analog to rat submaxillary gland.

The substance P (SP) analog [tyrosine1, norleucine11]-substance P [( Tyr1, Nle11]-SP) was iodinated by the chloramine-T method to yield the mono iodinated derivative; both peptides were biologically active on the guinea-pig ileum SP receptor. Saturable, reversible binding of [125l-Tyr1, Nle11]-SP to a single class of noninteracting sites on the rat submaxillary gland homogenate was demonstrated with an affinity of 9.26 +/- 0.8 nM and a maximum binding of 15.7 +/- 1.25 or 250 +/- 20 fmol/mg of protein. The relative potencies of SP and its fragments SP(2-11) and SP(4-11), as measured by their IC50, are in agreement with their rank order in the salivation assay, whereas physalaemin was found apparently weaker than expected. However, their rank order in displacing [125I-Tyr1, Nle11]-SP was identical to their rank order in displacing [125I-Bolton-Hunter]-SP bound to mouse mesencephalic cells in primary culture.

Animals↗
PubMed1983

Failure of opiates to increase the hydrolysis of GTP in neuroblastoma-glioma 108-15 cells.

It has been repeatedly demonstrated that the neuroblastoma-glioma (NG 108-15) cell line has opiate receptors that inhibit adenylate cyclase and it has been proposed that this inhibition is mediated by a naloxone reversible stimulation of a low Km GTPase (Koski and Klee, Proc. Natl. Acad. Sci. 78:4185, 1981). The guanine nucleotides of NG cells were labeled with [3H]guanine followed by incubation with 10(-6)M guanine. Etorphine (10(-6)M) or vehicle were added and the incubations continued for 1-4 min. The reaction was stopped with 5 percent TCA containing nucleotides as carriers and markers for the HPLC. Marker nucleotides were detected at 254 nm and the labeled nucleotides by liquid scintillation spectrometry. In several experiments, etorphine failed to produce any measurable change in the labeled nucleotides or in the GTP/GDP ratios. To verify that the opiate receptors were functional we measured its capacity to inhibit the formation of cAMP induced by PGE1. We also studied the effects of naloxone and PGE1 on the formation of cAMP in opiate tolerant cells. Tolerant cells responded to naloxone with a 50 percent increase in cAMP, indicating again that the opiate receptors were functional. Our results are consistent with the idea that in intact NG108-15 cells the opiate-mediated hydrolysis of GTP observed in cell membrane preparations is of very small magnitude.

Alprostadil↗
PubMed1983

The determination of dissociation constants for substance P and substance P analogues in the guinea pig ileum by pharmacological procedures.

The dissociation constants (Kd values) of substance P (SP), physalaemin, kassinin, and SP analogues acting on SP receptors in guinea pig ileal longitudinal muscle strips were determined by the pharmacological procedures of Furchgott [Adv. Drug Res. 3:21-55 (1966)]. This method involves analysis of the concentration-response data before and after fractional inactivation of receptors with phenoxybenzamine (2 X 10(-5) M). Estimations of the Kd values for SP were similar when phenoxybenzamine was incubated for 10, 13, or 15 min. Coincubation with high concentrations of SP protected against receptor inactivation with phenoxybenzamine, but bradykinin and serotonin did not cross-protect SP receptors. Kd values for SP were similar when trypsin was substituted for phenoxybenzamine [Kd = 8.1 +/- 4 nM (n = 9) versus 10 +/- 6 nM (n = 5)]. In atropinized preparations the Kd values obtained for physalaemin were similar to those obtained for untreated preparations [Kd = 8.0 +/- 3.6 nM (n = 5) and 12.6 +/- 3 nM (n = 4), respectively]. The effects of phenoxybenzamine on concentration-response curves for kassinin showed greater shifts to the right with phenoxybenzamine. This indicated that kassinin may interact with another population of receptors, in addition to the sites that SP and other analogues bind. A direct correlation was found between EC50 values and Kd values and Kd values for SP and SP analogues. It was estimated that, for SP, a 20% receptor occupancy is required to elicit a 50% response.

Animals↗
PubMed1983

High-affinity dextromethorphan binding sites in guinea pig brain. I. Initial characterization.

Tritiated dextromethorphan ([3H]DM) binds to two distinct sites in guinea pig brain, a high-affinity site (Kd = 13-20 nM) and a low-affinity site (Kd greater than 200 nM). Binding of [3H] DM to the high-affinity site is rapid, reversible, saturable, proportional to tissue concentration, and pH-dependent. The sites have a protein-like component, since preincubating brain homogenate in the presence of proteolytic enzymes and protein-modifying reagents significantly reduces binding. There is also a progressive loss of binding when brain homogenate is heated to temperatures in excess of 37 degrees. Millimolar concentrations of lithium, calcium, magnesium, and manganese decrease DM binding while sodium, in concentrations as high as 100 mM, has little effect; calcium in micromolar concentrations slightly enhances binding. The pons-medulla and cerebellum contain the highest density of sites. Subcellular localization studies have shown that high-affinity sites are confined almost exclusively to the microsomal fraction. Binding of DM to brain microsomes does not appear to be related to drug-metabolizing enzymes. The characteristics of DM binding suggest that DM sites are not a subclass of opiate receptors. Studies using tritiated dextrorphan as radioligand failed to reveal a high-affinity binding site for in brain.

Animals↗
PubMed1983

High-affinity dextromethorphan binding sites in guinea pig brain. II. Competition experiments.

Binding of dextromethorphan (DM) to guinea pig brain is stereoselective, since levomethorphan is 20 times weaker than DM in competing for DM sites. In general, opiate agonists and antagonists as well as their corresponding dextrorotatory isomers are weak competitors for tritiated dextromethorphan ([3H]DM) binding sites and display IC50 values in the micromolar range. In contrast, several non-narcotic, centrally acting antitussives are inhibitory in the nanomolar range (IC50 values for caramiphen, carbetapentane, dimethoxanate, and pipazethate are 25 nM, 9 nM, 41 nM, and 190 nM, respectively). Other antitussives, such as levopropoxyphene, chlophedianol, and fominoben, have poor affinity for DM sites whereas the antitussive noscapine enhances DM binding by increasing the affinity of DM for its central binding sites. Additional competition studies indicate that there is no correlation of DM binding with any of the known or putative neurotransmitters in the central nervous system. DM binding is also not related to tricyclic antidepressant binding sites or biogenic amine uptake sites. However, certain phenothiazine neuroleptics and typical and atypical antidepressants inhibit binding with IC50 values in the nanomolar range. Moreover, the anticonvulsant drug diphenylhydantoin enhances DM binding in a manner similar to that of noscapine. Preliminary experiments utilizing acid extracts of brain have not demonstrated the presence of an endogenous ligand for DM sites. The binding characteristics of DM sites studied in rat and mouse brain indicate that the relative potencies of several antitussives to inhibit specific DM binding vary according to species. High-affinity, saturable, and stereoselective [3H]DM binding sites are present in liver homogenates, but several differences have been found for these peripheral binding sites and those described for brain. Although the nature of central DM binding sites is not known, the potent interaction of several classes of centrally acting antitussives with DM sites suggests that they may be related to the mechanism of action of this drug.

Animals↗
PubMed1982

Hydrolysis of substance P and bradykinin by black widow spider venom gland extract.

Black widow spider venom gland extract was found to contain significant peptidase activity. Aliquots of the venom gland extract incubated at 37 degrees inactivated substance P (SP) and bradykinin but not angiotensin II or the enkephalins. The peptide inactivation was proportional to the duration of the incubation and the amount of extract used. Analysis of the peptides on high pressure liquid chromatography demonstrated that the loss in biological activity of SP and bradykinin in the longitudinal muscle of the guinea pig ileum was correlated with cleavage of the peptides into several fragments. Kinetic studies revealed that SP was initially split into two fragments but that these products underwent further degradation into smaller peptides. The optimal pH for the peptidase activity was 6.5. At 0 degree the enzymatic activity was undetectable, and it was irreversibly destroyed by incubation at 100 degrees for 5 min or by pretreatment of the extract with 100 microM diisopropyl fluorophosphate. In addition, the gland extract preparation hydrolyzed artificial substrates designed to detect trypsin or chymotrypsin-like activity.

Animals↗
PubMed1982

Fading and tachyphylaxis to the contractile effects of substance P in the guinea-pig ileum.

Substance P (SP) caused an immediate and vigorous contraction of the longitudinal smooth muscle layer of the guinea-pig ileum. The contractile response to SP, unlike that to acetylcholine or histamine was not maintained but faded to baseline levels in about 6 min. When 0.3-1.0 nM SP was added the fading time was shorter than 6 min and tachyphylaxis did not develop. Higher concentrations of SP produced fading times of about 6 min that could not be increased even by adding extremely high concentrations of the peptide, up to 1800 nM. Short fading times and the lack of development of tachyphylaxis are the result of the rapid adsorption and/or metabolism of SP. The addition of exogenous peptidases such as pronase, chymotrypsin and an extract of black widow spider venom gland dramatically increased the rate of degradation of SP, shortened the fading response and blocked the development of tachyphylaxis. Tetrodotoxin and atropine reduced the fading time by 25%, while eserine increased its duration several-fold; these findings are consistent with the existence of a cholinergic nerve component in the mediation of some of the effects of SP receptor and, in part, to adsorption and metabolism of the peptide. The magnitude of the tachyphylaxis to SP was proportional to the concentration of the desensitizing dose of the peptide and was specific to SP and to the related peptide physalaemin; no cross-tachyphylaxis towards other agents was found.

Animals↗
PubMed1981

Naloxone reversal of insulin-induced hypotension in reserpine pretreated rats.

The administration of insulin caused a gradual lowering of systolic and diastolic blood pressure in anaesthetized reserpinized rats. Injection of 1 mg/kg naloxone at the peak of the hypotension resulted in immediate restoration of blood pressure to pre-insulin control values. The recovery of the arterial blood pressure caused by naloxone lasted for 5 to 10 min and was entirely dependent on the reserpine pretreatment. The lowering of the blood pressure caused by insulin and the increase in systemic blood pressure after naloxone were of about the same magnitude in rats with bilateral denervation of the adrenal glands as in sham operated rats. It is concluded that in anaesthetized reserpinized rats, hypoglycemia causes the release of opiate-like material that mediates a hypotensive response. The origin, nature and site of action of this opioid activity is as yet not established, but does not appear to derive from the adrenal gland.

Adrenal Glands↗
PubMed1978

Electrically induced opiate-like inhibition of the guinea-pig ileum: cross-tolerance to morphine.

The guinea-pig myenteric plexus-longitudinal muscle (MPLM) preparation electrically stimulated at 0.1 Hz is very sensitive to the inhibitory effects of opiates. We used this preparation to detect an inhibitory response (IR) which was produced by electrical stimulation at 5 to 20 Hz. The magnitude and duration of the IR are determined by the parameters of the stimulation, mainly by the frequency and duration of the period of stimulation. Maximal IR is obtained with symmetrical biphasic stimuli of 2 msec duration and supramaximal voltage at 20 Hz applied for a period of about 5 min. The IR is calcium-dependent, cannot be attenuated by washing and is mediated by several components. About 55 to 70% of the IR can be reversed by specific narcotic antagonists and therefore it is considered to be produced by the release of endorphins. There are at least two additional components, one small, adrenergic in nature, and a third one which has not been identified. The offset rate of the IR is measured in minutes, while enkephalin and human beta-endorphin have half-lives of 10 and 85 sec, respectively, after washing. This suggests that the endorphin that mediates the opiate component of the IR may be a different one with slower offset rates. Myenteric plexus-longitudinal muscle strips obtained from guinea pigs which were made tolerant to morphine by subcutaneous implantation of three pellets were cross-tolerant to the opiate component of the IR elicited by electrical stimulation.

Animals↗
PubMed1977

Endogenous opiate receptor ligand: electrically induced release in the guinea pig ileum.

Opiate receptors mediate the electrically evoked inhibition of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. The electrically induced activation of the opiate receptor was produced by a prolonged simulation at 10 hertz and provides the first evidence that an endogenous opiate receptor ligand is released by nerve stimulation. The specificity of the phenomenon was demonstrated by the reversal obtained with the narcotic antagonists naloxone, naltrexone, and GPA 1843; GPA 1847, the (+)-isomer of 1843, did not cause reversal. The model system described should be useful for the study of the storage, synthesis, and release of endorphins.

Electric Stimulation↗
PubMed1977

The effect of enkephalin and enkephalin analogs on the guinea-pig ileum and rat brain opilate receptor.

We have synthesized the two naturally occurring enkephalins (Methionine-Enkephalin and Leucine-Enkephalin) as well as four other analogs (Methinonine-Enkephalin amide, S-Benzyl sulphonium analog of Methionine-Enkephalin, 3-Benzyl-tyrosine analog of Methionine-Enkephalin and 3-Benzyl-tyrosine analog of Leucine-Enkephalin) in order to study their relative potency and their inactivation of different tissues. Morphine-like activity of the peptides was determined by measuring their inhibitory effect in the guinea-pig ileum and their interaction with the brain opiate receptor; the order of affinity of the three most active peptides (Methionine-Enkephalin amide greater than Methionine-Enkephalin greater than Leucine-Enkephalin) for the rat brain opiate receptor was found to be the same in the guinea-pig ileum. The rate of inactivation of the peptides was also studied in the guinea-pig ileum and in the rat brain membrane preparation. Methionine-Enkephalin amide was relatively more resistant to the ileum peptidases than the other peptides, but was destroyed at the same rate by brain membranes; this variation in inactivation of Methionine-Enkephalin amide probably indicates the presence of differnet peptidases in each tissue preparation.

Animals↗