Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.     

A fragment of substance P with specific central activity: SP(1-7)

Amino-terminal fragments of substance P (SP), SP(1-7) and SP(1-8), were found to produce naloxone-reversible antinociception in the mouse similar to that produced by SP. Similar to SP, these peptides produce antinociception only within a narrow dose range. They have no activity on smooth muscle or blood pressure. These results suggest that contrary to peripheral effects of SP, which are mediated by receptors which recognize the carboxy-terminal part of the SP molecule, certain central actions of SP are mediated by receptors which recognize the amino-terminal part of the SP molecule. SP may be metabolized to this active fragment prior to its action at these receptors.     

Anabolic-androgenic steroids affect the content of substance P and substance P(1-7) in the rat brain

The effects of intramuscular (i.m.) injections of nandrolone decanoate (15 mg/kg/day), an anabolic-androgenic steroid, on the levels of substance P (SP) and on its N-terminal fragment SP(1-7) were examined in the male rat brain by radioimmunoassay. The results demonstrated that the SP immunoreactivity in amygdala, hypothalamus, striatum, and periaqueductal gray was significantly enhanced, whereas the concentration of the N-terminal fragment SP(1-7) was enhanced in the nucleus accumbens and in periaqueductal gray. In the striatum the steroid induced a decrease in the content of SP(1-7). The relevance of these peptides in connection with anabolic-androgenic steroid-induced aggression is discussed.     

Distinct binding sites for substance P and neurokinin A (substance K): co-transmitters in rat brain

The distribution of binding sites in rat brain for iodinated neurokinin A and iodinated substance P were compared using autoradiography. Distinct patterns of binding for the two iodinated tachykinins were noted. Binding sites for iodinated neurokinin A were noted in the olfactory bulb, cortex, supraoptic n., paraventricular n., certain amygdaloid n., hippocampus, medial habenula, interpeduncular n., n. of the tractus solitarius, and dorsal horn of the spinal cord. This pattern was in contrast to low levels of binding of iodinated substance P to the cortex, supraoptic n., paraventricular n., and the interpeduncular n., but substantial density of binding sites in numerous other regions.     

Anxiolytic-like effects of substance P fragment (SP(1-7)) in non-human primates (Callithrix penicillata)

The behavioral effects of the amino (N)-terminal fragment of substance P (SP(1-7)) on the marmoset (Callithrix penicillata) predator confrontation test of fear/anxiety were investigated. The test apparatus consisted of a figure-eight maze with three parallel arms interconnected at each extremity to a perpendicular arm. A taxidermized oncilla cat (Felis tigrina) was placed outside the maze facing one of its corners. Subjects were submitted to seven 30 min maze habituation trials (HTs), in the absence of the 'predator', and then to six 30 min treatment trials (TTs), in the presence of the 'predator', consisting of four doses of SP(1-7) (5, 50, 250 and 500 microg/kg; IP), saline and sham injection. SP(1-7) treatment reversed, in a dose-dependent way, the fear-induced avoidance behavior due to the predator's presence and increased the frequency of exploratory behaviors. Locomotor activity decreased during successive HTs, yet increased after all SP(1-7) treatments. These results indicate that systemic administration of SP(1-7) produces anxiolytic-like effects in marmosets tested in the predator confrontation model of fear/anxiety.     

Binding studies of substance P anterior pituitary binding sites: changes in substance P binding sites during the rat estrous cycle

Previous studies have shown that substance P (SP), an undecapeptide widely distributed in the gastrointestinal tract and in the peripheral and central nervous system, is a putative regulatory peptide involved in the control of reproductive function. Specifically, SP inhibited, at the anterior pituitary (AP) level, the stimulatory action of a physiological concentration (10(-8) M) of Gonadotropin Releasing Hormone (GnRH) on the release of the luteinizing hormone (LH). In the present work, we have demonstrated the presence of specific SP binding sites in the AP and related changes in the number of these sites to GnRH receptor number, hypothalamic SP and GnRH content and LH secretion during the rat estrous cycle. High affinity saturable SP binding sites (Kd, 1.5 approximately equal to 10 nM) were demonstrated in AP membranes using [3H]-SP or a novel analog, [125I]-(D-Tyr0, NorLeu11)SP. The binding affinity of SP fragments decreased with progressive removal of amino acid residues from N or C termini of the molecule. Other neuropeptides had low affinity for the SP binding sites. During the rat estrous cycle, SP and GnRH binding capacity of the anterior pituitary were inversely related. At the time of the proestrous LH surge, the AP binding capacity was low for GnRH but high for SP. The highest content of SP in the hypothalamus were recorded during the afternoon of proestrus when hypothalamic GnRH levels were lowest and the preovulatory surge occurred. These studies have established the presence of high affinity specific binding sites for SP in the AP which alter during the estrous cycle in a manner appropriate for mediating the direct inhibitory effects of SP on LH release in vitro.     

Light microscopic autoradiographic localization of [3H] substance P binding sites in rat thoracic spinal cord

We have used light microscopic autoradiography to look for the distribution of [³H] substance P receptors in the thoracic spinal cord of the rat. High densities of autoradiographic grains were localized to the intermedialateral cell column, the central canal and the substantia gelatinosa of the dorsal horn.     

Comparative distribution of substance P (SP) and cholecystokinin (CCK) binding sites and immunoreactivity in the brain of the sea bass (Dicentrarchus labrax).

Specific binding sites for cholecystokinin (CCK) and substance P (SP) were detected in the brain of a marine teleost fish, the sea bass, after in vitro incubation of tissue sections with the tritiated peptides and light microscopic autoradiography. Specific binding sites for [3H]-CCK were detected in the dorsal and ventral telencephalon, in the preoptic, tuberal and posterior hypothalamus, in the optic tectum, in the valvulla cerebelli, in the vagal lobe and further in a dorsal location in the medulla oblongata. Areas rich in [3H]-SP binding were located in the ventral telencephalon, in the entire hypothalamic and thalamic region, in the midbrain tegmentum, in the optic tectum, in the valvulla cerebelli and in the medulla oblongata. The distribution of these binding sites seemed to match fairly well with the location of the corresponding immunoreactive elements, although some minor mismatches could be observed. These autoradiographic findings provide the first anatomical evidence for the presence of CCK-like and SP-like binding sites in the brain of a teleost fish.     

Endomorphin-2, deltorphin II and their analogs suppress formalin-induced nociception and c-Fos expression in the rat spinal cord

In this study, we evaluated the effects of intrathecally administered agonists of mu- and delta-opioid receptor and their analogs on the pain-induced behavior and expression of c-Fos immunoreactivity in the spinal cord, elicited by intraplantar injection of 12% formalin to the hindpaw of the rat. Previous report from our laboratory and other author's study indicated that intrathecal administration of mu agonists morphine and endomorphin-2 and delta-opioid agonist deltorphin II produced a dose-dependent antinociceptive effects in acute and inflammatory pain. In this study, intrathecal injection of morphine (10 microg), endomorphin-2 (5 microg) and its analog Dmt-endomorphin-2 (10 microg) significantly decreased the formalin-induced pain behavior, and lowered a number of c-Fos positive neurons in the laminae I, II and III of the spinal cord by about 40%, 30% and 40%, respectively. Significant reduction of formalin-induced behavioral responses was also observed after i.th. administration of deltorphin II (15 microg) and its analog ile-deltorphin II (15 microg). Agonists of delta-opioid receptor significantly reduced a number of c-Fos positive neurons by about 28% and 40%, respectively. Analog of endomorphin-2 and analog of deltorphin II suppressed more potently expression of c-Fos in the dorsal horn of the spinal cord than the parent peptides. Our study indicates that new analogs of mu- and delta-opioid receptor exhibit strong antinociceptive potency similar or even higher than the parent peptides, and that their effect is positively correlated with the inhibition of c-Fos expression.     

Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 microns paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.     

Interactions of substance P antagonists with serotonin in the mouse spinal cord

Administered intrathecally (IT) to mice, the putative substance P antagonist [D-Pro2,D-Trp7,9-substance P (DPDT) blocked substance P- and serotonin-induced reciprocal hindlimb scratching with ID50 values of 4.6 (2.9-6.9) and 3.0 (1.9-4.8) micrograms, respectively. The duration of this antagonistic effect was 90-120 min. In contrast, DPDT did not block bombesin-, somatostatin-, glycine- or glutamate-induced scratching. These data indicate that DPDT is an effective antagonist of serotonin-induced behaviors in the mouse spinal cord. Phenoxybenzamine (IT) also blocked substance P- and serotonin-induced scratching. Its onset of action was more rapid for serotonin than for DPDT implying differences in agonist-induced receptor activation. Methysergide (IT) blocked serotonin-induced scratching [ID50 = 0.7 (0.3-1.5) micrograms], but not substance P-induced scratching. Similar to DPDT, [D-Arg1,D-Trp7,9,Leu11]-substance P, [des-Arg1,D-Pro2, D-Trp7,9]-substance P(2-11) and [D-Pro4,D-Trp7,9]-substance P(4-11) blocked substance P and serotonin-induced scratching. In contrast, [D-Pro2,D-Phe7,D-Trp9]-substance P and [D-Pro4,D-Trp7,9,10]-substance P(4-11) blocked only substance P-induced scratching. Thus, some, but not all putative substance P antagonists may also be behavioral antagonists of serotonin in the mouse spinal cord.     

Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

Summary At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 m paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.The work, presented at the Histochemical Society's 29 Annual Meeting in Vancouver, B.C. April 1–2, 1978, was partially supported by CCHD 10-12-04-3600-67 (LLV)     

Specific substance P binding sites in rat thymus and spleen: in vitro autoradiographic study

Substance P binding sites were localized in rat thymus and spleen by incubation of tissue sections with [125I]Bolton-Hunter substance P, [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry and comparison with 125I standards. The tissue localization of the binding sites was determined with emulsion autoradiography. A single type of specific, saturable, high affinity binding sites was found associated with the vasculature in the medulla of the thymus and the marginal sinus of the spleen, with a Kd of 0.10 and 0.14 nM, respectively. Of all the unlabeled tachykinins tested (substance P, physalaemin, substance K, eledoisin, kassinin, and neuromedin K) substance P was the most potent inhibitor of [125I]Bolton-Hunter substance P binding, with an IC50 of approximately 0.5 nM, indicating the presence of substance P-P binding sites. Our results support the hypothesis of a role for substance P in the modulation of the immune system.     

Osmotic regulation of substance P and neurokinin A peptide content and substance P binding sites in distinct hypothalamic nuclei of the rat.

Quantitative receptor autoradiography using Bolton-Hunter iodinated substance P (SP) was used to localize specific sites in the rat hypothalamus. The amount of SP and neurokinin A (NkA) in extracts from discrete areas of the hypothalamus was measured using specific radioimmunoassays. A high density of SP binding sites was observed in the perimeter of the magnocellular paraventricular and supraoptic nuclei, while the magnocellular nuclei themselves possessed a low receptor density. In control animals, the number of SP binding sites was also low in the arcuate nucleus and the median eminence. Substance P and NkA peptide concentrations were highest in the paraventricular nucleus (PVN), decreasing in the following order: arcuate nucleus (Arc) greater than median eminence (ME) greater than supraoptic nucleus (SON) greater than subfornical organ (SFO). In animals given 340 mmol/l NaCl instead of tap water to drink for 12 days, significant increases in the number of SP binding sites occurred in the medial parvocellular subdivision of the PVN, periamygdaloid cortex, medial preoptic nucleus, Arc, and ME, but other hypothalamic areas were unaffected. In saline-treated animals, significant increases in SP and NkA peptide concentrations were observed in the ME, while in the SFO only the concentration of NkA increased significantly. In the SON, substance P and neurokinin A levels were doubled, whereas in the PVN and Arc no changes in peptide levels were observed. Chronic osmotic stimulation is associated with lowered circulating levels of adrenocorticotropin releasing hormone (ACTH), and the present data further substantiate the hypothesis that hypothalamic tachykinin-containing neuronal terminals are centrally involved in the inhibition of anterior pituitary ACTH release observed during chronic osmotic stimulation.     

Characterization of the [125I]endomorphin-2 binding sites in the MCF7 breast cancer cell line

In the present study, the expression of the micro-opioid receptor on protein level has been demonstrated in MCF7 breast cancer cells. Binding of the [125I]-labeled micro-opioid receptor selective ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) was examined in vitro using a cross-linking assay followed by a Western blot technique. The radioactive complex had a molecular weight of about 65 kDa and was detectable by anti-micro-opioid receptor antibody, indicating the presence of micro-opioid receptors in MCF7 cell membranes. Characterization of endomorphin-2 binding to the membranes obtained from MCF7 cells was performed. Cold saturation experiments with [125I]endomorphin-2 showed biphasic binding curves in Scatchard coordinates. One component represents a high affinity and low capacity, and the other low affinity and higher capacity binding sites. The obtained Bmax values for [125I]endomorphin-2 binding to MCF7 membranes were much higher than those obtained for mouse brain. Pharmacological characterization of the [125I]endomorphin-2 binding sites was made using endomorphin-2 and two other micro selective ligands, morphiceptin, and [D-1-Nal3]morphiceptin on MCF7 cell membrane preparations and whole MCF7 cells. In both cases, the rank order of potency was [D-1-Nal3]morphiceptin>endomorphin-2>morphiceptin, but in case of whole MCF7 cells the IC50 values were about 40 times higher.