Inability of [125I]Sar1, Ile8-Angiotensin II to Move Between the Blood and Cerebrospinal Fluid Compartments

The angiotensin II competitive antagonist [125I]-Sar1, Ile8-angiotensin II was not transported from the vascular space to the cerebroventricular space in either intact or nephrectomized rats. In addition [125I]Sar1, Ile8-angiotensin II lacked the capacity to move in the opposite direction over a 20-min collection period following cerebroventricular infusion. These data suggest that angiotensins lack the capacity to move freely between the blood and cerebrospinal fluid compartments and are consistent with the notion that blood-borne and cerebroventricular angiotensins access different receptor populations.     

Evidence for angiotensin II receptors in the urinary bladder of the rabbit

Dose-response (DR) curves for several angiotensin analogs were examined on isolated rabbit detrusor strips with washout and rest between each addition. The order of potency was [Val5]-angiotensin II greater than [Ile5]-angiotensin II greater than [Ile5]-angiotensin I greater than [Val4]-angiotensin III. Repeated cumulative DR to [Val5]-AII resulted in a gradual increase in potency and intrinsic activity for four DR. However, the maximum force generated occurred at lower agonist concentrations and was less than that of the single methods, suggesting tachyphylaxis. Atropine (1.0 microM) shifted the cumulative DR curve downward, suggesting some cholinergic component possibly involving a presynaptic site of action. The magnitude of field-stimulated atropine-resistant contractions was reduced by both 1.0 and 10 microM saralasin as well as 10 microM naloxone. Tissue binding with 125I-labelled angiotensin II on isolated detrusor smooth muscle membranes indicated specific binding saturation occurred at 14.3 fmol/mg with a KD of 0.72 nM in EDTA-Tris buffered saline. Thus our results show that angiotensin II (AII) receptors can be demonstrated in destrusor muscle by ligand binding experiments on cell membranes and that saralasin and naloxone partially block atropine-resistant contractions. However, it seems unlikely that AII serves as a neurotransmitter because of the delay in onset of action of exogenous AII in isolated bath experiments and the apparent inability of saralasin to totally abolish the atropine-resistant field-stimulated preparation. If AII serves a role in neurotransmission it most probably is as a neuromodulator.     

Partial agonistic effect of 9-hydroxycorynantheidine on mu-opioid receptor in the guinea-pig ileum

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that is reported to have opioid agonistic properties. The 9-demethyl analogue of mitragynine, 9-hydroxycorynantheidine, is synthesized from mitragynine. 9-Hydroxycorynantheidine inhibited electrically stimulated guinea-pig ileum contraction, but its maximum inhibition was weaker than that of mitragynine and its effect was antagonized by naloxone, suggesting that 9-hydroxycorynantheidine possesses partial agonist properties on opioid receptors. Receptor binding assays revealed that 9-hydroxycorynantheidine has high affinity for mu-opioid receptors. In an assay of the guinea-pig ileum, naloxone shifted the concentration-response curves for [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), (5alpha,7alpha,8beta)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) and 9-hydroxycorynantheidine to the right in a competitive manner. The pA(2) values of naloxone against 9-hydroxycorynantheidine and DAMGO were very similar, but not that against U69593. As indicated by the two assay systems, the opioid effect of 9-hydroxycorynantheidine is selective for the mu-opioid receptor. 9-Hydroxycorynantheidine shifted the concentration-response curve for DAMGO slightly to the right. Pretreatment with the mu-opioid selective and irreversible antagonist beta-funaltorexamine hydrochloride (beta-FNA) shifted the concentration-response curve for DAMGO to the right without affecting the maximum response. On the other hand, beta-FNA did not affect the curve for 9-hydroxycorynantheidine, but decreased the maximum response because of the lack of spare receptors. These studies suggest that 9-hydroxycorynantheidine has partial agonist properties on mu-opioid receptors in the guinea-pig ileum.     

Activation of angiotensin II receptors in brain potentiates the stimulating effect of endogenous opioid neurons on central sympathetic outflow

Endogenous opioid peptides appear to have neurotransmitter or neuromodulator functions in brain mediating a wide variety of effects. We have reported that intracisternal administration of synthetic human beta-endorphin increases plasma concentration of catecholamines, apparently by acting at unknown brain sites to increase sympathetic outflow to the adrenal medulla and sympathetic nerves. In the present study we examined the possibility that angiotensin II, acting in brain, modulates endorphin-induced catecholamine secretion. Simultaneous intracisternal administration of angiotensin II 1.0 nmol together with synthetic human beta-endorphin 1.45 nmol potentiated the plasma epinephrine, norepinephrine and dopamine responses to intracisternal beta-endorphin. In contrast, simultaneous intracisternal administration of the angiotensin II antagonist, [Sar1, Val5, Ala8]-angiotensin II (saralasin), 1.1 nmol together with beta-endorphin, blunted the plasma epinephrine, norepinephrine and dopamine responses to beta-endorphin. These data are consistent with the hypothesis that activation of angiotensin II receptors in brain potentiates the endorphin-induced stimulation of central sympathetic outflow. It remains to be demonstrated whether angiotensin II acting in brain to modulate activity of opioid neurons is synthesized in brain or is derived peripherally.     

Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.     

The role of mu- and delta-opiate receptors in the realization of the autonomic effects of opioid peptides

In the first series of experiments on the isolated mouse vas deferens and guinea-pig ileum the capacity of 10 opioid peptides to activate mu- and delta-receptors was evaluated. [DAla2, DLeu5]-enkephalin (DADLE) and [DAla2, MePhe4, Gly5-ol]-enkephalin (DAMPGE) were the most selective agonists of delta- and mu-opiate receptors, respectively. In the second series of experiments on urethan-anesthetized rats it was shown, that intravenous administration of DADLE or DAMPGE (10(-7) M/kg each) elicited hypotension, bradycardia and expiratory apnoe. These effects disappeared both after naloxone injection and bilateral cervical vagotomy. A reflex nature of the vegetative effects of opioid peptides and the role of both mu- and delta-receptors in their realization are suggested.     

A chimeric opioid peptide with mixed mu agonist/delta antagonist properties.

There is evidence to indicate that opioid compounds with mixed mu agonist/delta antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective mu agonist H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) (Dmt=2',6'-dimethyltyrosine) and the potent and selective delta antagonist H-Tyr-TicPsi[CH2-NH]Cha-Phe-OH (TICP[Psi]) (Cha=cyclohexylalanine), connected 'tail-to-tail' via a short linker, was synthesized using a combination of solid-phase and solution techniques. The resulting peptide, H-Dmt-->D-Arg-->Phe-->Lys-NH-CH2-CH2-NH-Phe<--Cha[NH-CH2]PsiTic<--Tyr-H, showed the expected mu agonist/delta antagonist profile in the guinea-pig ileum and mouse vas deferens assays. Its mu and delta receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.     

Presynaptic modulation by noradrenaline and an opioid of the substance P-induced release of [3H]acetylcholine from the myenteric plexus

Substance P (7.5-750 nM) applied in superfusion dose-dependently released 3H from isolated strips of myenteric plexus-longitudinal muscle of the guinea-pig ileum loaded with [3H]choline. Separation of the [3H]acetylcholine and [3H]choline components of the released radioactivity revealed that in response to substance P (SP) administration only the release of [3H]acetylcholine increased above resting level. A slowly developing tachyphylaxis to the effect of SP was observed. Evidence has been obtained that the slow tachyphylaxis developed to the acetylcholine-releasing effect of SP was not due to the exhaustion of releasable acetylcholine pool. Release of acetylcholine by 150 nM SP was completely prevented by tetrodotoxin or in a Ca2+-free medium and greatly reduced in the presence of noradrenaline or the opioid receptor agonist (D-Met2,Pro5)-enkephalinamide. The effect of noradrenaline and the opioid peptide was apparently prevented by yohimbine and naloxone, respectively.     

Endogenous ligands for sigma opioid receptors in the brain ("sigmaphin"): evidence from binding assays

Two endogenous ligands which interact preferentially with the sigma opioid receptors were identified from the guinea-pig brain extract in a Sephadex G-50 fractionation. These two ligands inhibited more potently the binding of [3H]SKF-10047 to sigma opioid receptors than [3H]naloxone to mu opioid receptors, [3H]ethylketocyclazocine to kappa opioid receptors and [3H]DADLE to delta opioid receptors. In the phencyclidine receptor assay, these two ligands were almost inactive. Incubation of these ligands with trypsin destroyed at least 50% of the activities in the sigma opioid receptor assay. Both ligands inhibited the sigma binding in a dose-dependent manner. The inhibition could be eliminated when the two ligands were removed from incubation media by extensive washings. It is therefore concluded that sigma opioid receptors are not phencyclidine receptors and that endogenous ligands for sigma opioid receptors may exist in the brain.     

Leumorphin is a novel endogenous opioid peptide in man

Porcine leumorphin, a putative opioid peptide corresponding to amino acid residues 228-256 of preproenkephalin B has been demonstrated to exist in the porcine neurointermediate pituitary. A recent study on the sequence analysis of genomic DNA of human preproenkephalin B has shown that human leumorphin differs in 3 amino acid residues from porcine leumorphin. In order to clarify whether leumorphin is an endogenous opioid peptide in man, we have studied its existence in the human brain using a radioimmunoassay and its opioid activity by a bioassay with the guinea-pig ileum myenteric plexus-longitudinal muscle preparation. High performance gel permeation chromatography and reverse-phase high performance liquid chromatography coupled with the radioimmunoassay for leumorphin have revealed that human leumorphin exists in water extracts of the human striatum. In the guinea-pig ileum assay, synthetic human leumorphin exhibited potent opioid activity, with the concentration of 3nM to give 50 per cent inhibition. These results indicate that leumorphin is a novel endogenous opioid peptide in man.     

Detection of a long acting endogenous opioid in blood and small intestine.

The blood of guinea-pigs and certain other species was found to contain two substances with opiate-like activity. These two substances could be separated by thin layer chromatography in a variety of solvent systems, which enabled them to be categorised as either fast or slow moving material. Although both substances caused a naloxone-antagonisable inhibition of the twitch tension of the electrically-stimulated myenteric plexus-longitudinal muscle strip from the guinea-pig ileum, the fast moving material differed in that its effect could only be reversed by many repeated washings of the preparation. Both fast and slow moving material were found to be 30 times less potent on the isolated mouse vas deferens than on the guinea-pig ileum preparation. The inhibiting effects of these opioids were not altered by incubation with either trypsin or pronase. An opioid was also detected in the fluid bathing strips of the guinea-pig ileum preparation. This opioid had similar properties to the fast moving material isolated from blood. The release of this material from strips of the guinea-pig ileum was not enhanced by electrical stimulation of the preparation.     

[3H]Diprenorphine Binding to k-Sites in Guinea-Pig and Rat Brain: Evidence for Apparent Heterogeneity

The binding of the unselective opioid antagonist [3H]diprenorphine to homogenates prepared from rat brain and from guinea-pig brain and cerebellum has been studied in HEPES buffer containing 10 mM Mg2+ ions. Sequential displacement of bound [3H]diprenorphine by ligands with selectivity for mu-, delta-, and kappa-opioid receptors uncovers the multiple components of binding. In the presence of cold ligands that occupy all mu-, delta-, and kappa-sites, opioid binding still remains. This binding represents 20% of total specific sites and is displaced by naloxone. The nature of these undefined opioid binding sites is discussed.     

Pyrazolo[3,4-d]pyrimidines as adenosine antagonists

A large number of nitrogen heterocycles structurally related to caffeine and theophylline have been tested for activity as adenosine antagonists. Preliminary screening, utilizing displacement of [3H]N6-phenylisopropyladenosine (PIA) binding to rat brain membranes, identified several pyrazolo[3,4-d]pyrimidines with potential antagonist activity. These were then tested for their ability to antagonize adenosine-stimulated adenylate cyclase of guinea-pig slices and to block adenosine receptors which mediate presynaptic inhibition of transmitter release from cholinergic nerves in guinea-pig ileum. Of several compounds found to have antagonist activity, one of these, 4,6-bis-alpha- carbamoylethylthio -1-phenylpyrazolo[3,4-d]pyrimidine ( DJB -KK) was approximately an order of magnitude more potent than theophylline in both tests. GTP greatly reduces the potency of purine agonists, but not antagonists, as inhibitors of [3H] PIA binding; the potency of the pyrazolo[3,4-d]pyrimidine compounds was not altered by GTP. The compounds have no significant activity against [3H]adenosine uptake or on the binding of ligands to muscarinic cholinergic, beta-adrenergic, GABA or L-glutamate receptors.     

Interaction of opioid peptides and the dopaminergic system in the myenteric plexus of the guniea pig ileum

The effects of treatment with dopamine agonists and 6-hydroxydopamine on the release of opioid peptides from the myenteric plexus of guinea-pig ileum were examined. Apomorphine or bromocriptine treatment at doses that act on dopamine autoreceptors to inhibit dopamine release resulted in a significant elevation of the release of opioid peptides. 6-hydroxydopamine treatment, which produces a lesion of catecholaminergic nerve terminals also resulted in an increase in opioid peptide release. These findings indicate that interruption of dopaminergic transmission in the myenteric plexus produces an increase in the release of opioid peptides and suggest an inhibitory modulation of opioid peptidergic neurons by dopamine systems in the myenteric plexus of the guinea-pig ileum.     

D-Ala2]deltorphin II analogs with high affinity and selectivity for delta-opioid receptor.

Nine [D-Ala2]deltorphin II (DL-II:Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2) analogs having various aliphatic amino acids at positions 5 and 6 were synthesized to gain more information about the role of hydrophobic Val5,6 residues for the delta-opioid receptor selectivity. Binding assays of analogs replaced by Ala demonstrated the importance of hydrophobic Val5,6 residues in DL-II for delta-affinity and selectivity, and especially critical importance of Val5 residue for higher delta-selectivity. By enhancing the hydrophobicity of residues at positions 5 and 6, we have developed analogs with very high delta-affinity and selectivity over those of DL-II, e.g., [Ile5,6], [norleucine5,6] and [gamma-methyl-leucine5,6]DL-II, which will be useful as delta-selective ligands for investigation of the physiological role of opioid receptors.     

Identification of 5HT2-Receptors on Longitudinal Muscle of the Guinea Pig Ileum

Abstract

In binding experiments with the radioligands [³H]Ketanserin (HKet) and [¹²⁵I]LSD (ILSD) 21 compounds were investigated using rat brain cortex membranes. The pK_D-values of the compounds were virtually independent of the radioligand used and their rank order was consistent with classification of the binding sites as being of the 5-HT₂-type. In contrast, in the longitudinal muscle of the guinea pig ileum in the presence of 0.3 μM cinanserin, ILSD labelled sites which were quite different to those in the cortex. In a functional test antagonism of the 5HT induced contraction of the guinea-pig ileum was measured in the presence of 1 μM atropine. The pharmacological inhibition constants (IC₅₀-values) of 8 compounds correlated well with the dissociation constants for HKet binding in the cortex and did not correlate with the data from ILSD binding in the guinea pig ileum. It is concluded that the ileum contains postjunctional 5HT₂-receptors which mediate contraction. The nature of the ILSD binding sites in the ileum remains to be elucidated.     

Effects of chronic treatment with atypical neuroleptics on the biosynthesis and release of opioid peptides in guinea-pig ileum

The guinea-pig ileum myenteric plexus is known to contain opioid peptides, which can be released by electric stimulation at high frequency. Haloperidol, a classic neuroleptic drug, increases the biosynthesis and release of endogenous opioid peptides from the myenteric plexus. In the present work we have examined the effects of chronic treatment with sulpiride and clozapine, two atypical neuroleptics, on the release of these peptides in the myenteric plexus of guinea-pig ileum. Both neuroleptics, administered over a period of 7 days, produced an increase of the inhibitory response obtained by electrical stimulation at 10 Hz of the ileum myenteric plexuslongitudinal muscle preparation. The inhibitory response was reversed by the specific opioid antagonist naloxone, which suggests that the increase in the inhibitory response produced by blocking the dopaminergic receptors is mediated by an increase in the release of opioid peptides. When sulpiride- or clozapine-treated guinea-pigs received cycloheximide (an inhibitor of peptide biosynthesis) there was a significant decrease of the inhibitory response, which indicates that neuroleptics produced an increase of the synthesis of opioid peptides in the ileum myenteric plexus. These results reveal a possible influence of the dopaminergic system on the biological turnover of these peptides.     

A new tachykinin receptor revealed by substance P analogues in the guinea pig ileum]

[Pro9]SP and septide have been described as selective agonists for the SP receptor (NK-1 type). These two peptides contract with a great efficacy the guinea-pig ileum, but unexpectedly septide was practically devoid of affinity for the NK-1 site labelled by 3H-[Pro9]SP. Like septide, SP analogues like SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP share the same peculiar properties. In addition, the contracting activity of these peptides is not explained by an interaction with NK-2 or NK-3 sites. GR 71,251, a compound which has been described as NK-1 antagonist, was more potent in inhibiting the septide- and the [Apa9-10]SP- than the [Pro9]SP-evoked contracting responses. Altogether, these results suggest that septide, SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP exert their high contracting activity in the guinea-pig ileum by acting on a new type of tachykinin receptor.     

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery.

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst1, msst2, msst3, msst4 and msst5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst1 and sst2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst4 and especially sst5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst1 and sst2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst2 in the periphery), sst3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst4 receptors are present) and it remains to be seen whether sst5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [125I]Tyr11-SRIF14 which in the presence of 120 mM labels primarily sst1 receptor as suggested by the better correlation hsst1 and similar rank order of potency. The profile of [125I]Tyr3-octreotide labelled sites in rat cortex correlates better with recombinant sst2 than sst3 or sst5 binding profiles. Finally, [125I]LTT-SRIF28-labelled sites in rat lung express a sst4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [125I]LTT-SRIF28, [125I]CGP 23996, [125I]Tyr10-CST, or [125I]Tyr3-octreotide. Although, [125I]Tyr10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [125I]Tyr3-octreotide represents a prominent part of the binding (when compared to [125I]LTT-SRIF28 and [125I]CGP 23996), suggesting that sst2 receptors are strongly expressed in most tissues; it is only in rat lung that [125I]LTT-SRIF28 and [125I]CGP 23996 show marked binding, whereas [125I]Tyr3-octreotide does apparently label no sites, in agreement with the sole presence of sst4 receptors in this tissue.     

Correlation between circular dichroism data and biological activities of 2,5 substituted enkephalin analogues

The relative structural rigidity of enkephalin analogues characterized by the molar ellipticity data obtained from the circular dichroism spectra of peptides was correlated with the opioid agonist activities of compounds displayed in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations. It was found that the so called μ receptors modelled by guinea-pig ileum preferred the analogues with high capacity to exist in folded form, whilst the so called δ receptors (mouse vas deferens) accepted flexible ligands as readily as rigid ones.