Characterization of 1,1-dimethyl-4-phenylpiperazinium induced increased proenkephalin processing in bovine chromaffin cells

Acute stimulation of bovine adrenal chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) gives rise to a significant increase in secretion of [Met5]-enkephalin immunoreactive material (ME-IRM) into the culture medium (1). Following this secretion the cellular ME-IRM levels do not decrease, suggesting the replenishment of the peptides. The repletion of the cellular ME-IRM appears to result from an increase in processing of large molecular weight peptides containing [Met5]-enkephalin and [Leu5]-enkephalin. Gel filtration chromatography on Bio-Gel P-10 was used to fractionate the enkephalin-like peptides (ELPs) present in the culture media and chromaffin cell extracts. Fractionation was done for samples before and after nicotinic receptor stimulation by DMPP to demonstrate the secretion and repletion of the ELPs. Gel chromatographic profiles of ELPs present in the culture media after DMPP stimulation revealed the presence of 4 peaks, representing different molecular forms of these peptides (Peaks 1-4), with a selective increase in secretion of Peaks 3 and 4. The chromatograms of ELPs extracted from cultured chromaffin cells showed similar patterns to those obtained from ELPs present in the culture medium after stimulation. Analyses of individual peaks after fractionation of cell culture extracts showed an increase in the amount of immunoreactive material found in Peak 4 with a concomitant decrease in the immunoreactivity found in the higher molecular weight peaks (Peaks 1-3). Further purification of Peak 4 from cell extracts on reversed-phase HPLC (RP-HPLC) showed a significant amount of ELPs existed as the sulfoxide derivative of [Met5]-enkephalin. The content of [Met5]-enkephalin sulfoxide (ME-O-enk) did not decrease following DMPP stimulation. We conclude that acute stimulation of nicotinic receptors in the chromaffin cells enhances the processing of proenkephalin precursors to keep pace with the secretion of low molecular weight peptides.     

Opioid peptides in adrenal gland

Enkephalin-like immunoreactive peptides have been observed in adrenal glands of all species studied with the highest contents found in dogs and cows, and the lowest in rats. These peptides are located both in gland cells and in afferent nerve terminals. Bovine adrenal glands contain opioid peptides in many molecular forms. The peptides include a group of low molecular weight forms (M.W. <1000) which are capable of binding to the opiate receptor, and a group of high molecular weight forms (M.W. >1000) which contain enkephalin within their peptide sequence, but are devoid of opioid activity unless treated with trypsin. The physiological role(s) of the adrenal enkephalin-like material is not clear at present. However, it has been observed that nicotine-stimulated release of catecholamine from isolated chromaffin cells can be reduced by opiate agonists, suggesting that enkephalin-like peptide in nerve terminals may act on chromaffin cells. Several lines of evidence suggest that enkephalin-like peptides in gland cells can be released into the bloodstream.     

Biochemical characterization of enkephalin-like immunoreactive peptides of adrenal glands

The total enkephalin-like immunoreactive peptide content of adrenal glands from dog, cattle, guinea pig and rat was investigated by radioimmunoassay using a (met⁵)-enkephalin antiserum. Dog adrenals contain the highest amount of peptides, cattle and guinea pig adrenals contain lesser amounts, and the rat adrenals had the least amount (0.05% that of the dog). Comparison of the (met⁵)-enkephalin content of the adrenal cortex and medulla with that of whole bovine adrenal gland indicates that the peptides are concentrated in the medulla. Analysis of the chromaffin granules from bovine adrenal medulla indicates this to be the primary storage site for (met⁵)-enkephalin-like peptides. Gel chromatography reveals a molecular heterogeneity of the immunoreactive peptides in all species tested; high molecular weight peptides account for a larger proportion of the immunoreactivity when compared with the low molecular weight peptides.     

Inhibitory effects of proopiomelanocortin on cortical and medullary activity in the calf adrenal.

The effects of the infusion of ACTH1-24 and proopiomelanocortin on the denervated adrenal gland have been investigated in conscious 3-6 weeks-old calves by means of the adrenal-clamp technique. To prevent variation in the release of endogenous ACTH the pituitary stalk was cauterized during preparatory surgery. ACTH1-24 (5 ng/min per kg, i.v.) increased the output of cortisol from the adrenal by about 500 ng/min per kg body weight and this effect was rapidly reduced by simultaneous infusion of ovine proopiomelanocortin at 5 ng/min per kg. Release of met5-enkephalin and leu-enkephalin from the adrenal was reduced by ACTH1-24 (P less than 0.05) and this effect was enhanced significantly by additional infusion of proopiomelanocortin (P less than 0.02). However met5-enkephalin represented less than one-third of the met-enkephalin containing peptide released and the output of this pool was unaffected by infusion of ACTH. Proopiomelanocortin had no effect on met5-enkephalin production nor that of the total pool of met5-enkephalin containing peptides. It is concluded that ACTH reduced the quantity of pro-enkephalin processed to met5-enkephalin.     

Extraction and Partial Characterization of Enkephalin-Like Peptides from Splanchnic Nerve

A method is described for the extraction of enkephalin-like peptides from peripheral nerve using chloroform and acidic methanol to facilitate a differential extraction of peptides and lipid. Porcine splanchnic nerve contains enkephalin-like peptides in low amounts compared to porcine adrenal medulla and striatum. Gel filtration chromatography reveals the presence of enkephalin-like peptides in both processed and cryptic forms. This is the first reported isolation and partial characterization of these peptides in splanchnic nerve. The presence of these peptides in this nerve provides support for the contention that the splanchnic nerve can modulate catecholamine release from the adrenal medulla through an effect on opiate receptors located on chromaffin cells.     

Further Characterization of an Enkephalin-Generating Enzyme from Adrenal Medullary Chromaffin Granules

An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.     

Enkephalin peptides in fetal sheep, changes with gestation, origin and production by the placenta.

Enkephalin-containing peptides have been followed in the circulation of fetal sheep between 118-143 days gestation. Using a combination of radioimmunoassay and hplc met5-enkephalin was found in the concentration range 60-500 pg/ml and proenkephalins containing met5-enkephalin had a concentration of 150-4000 pg/ml. The concentration of both increased towards term. The sources of the enkephalin peptides was investigated by measurement of differences across the umbilical circulation and by studying the effects of fetal adrenal demedullation and chemical sympathectomy. The placenta showed a continuous net output of enkephalin peptides which increased close to term. This placental output was increased sharply by reduction of uterine blood flow either using compression of the uterine artery or through infusion of adrenaline at 35 micrograms/min into the maternal circulation. Maternal hypoxia caused by breathing 9% O2 plus 3% CO2 also increased fetal plasma enkephalin levels, although not output from the placenta. Adrenal demedullation, particularly if accompanied by chemical sympathectomy depressed fetal plasma enkephalin concentrations and sharply suppressed the fetal peptide responses to maternal hypoxia. It is concluded that the placenta and the fetal adrenal are important sources of met5-enkephalin-containing peptides in the fetal circulation. The placental production appears to be closely tied to changes in uterine perfusion and adrenal output changes in response to fetal oxygenation.     

Adrenal vein catecholamines and neuropeptides during splanchnic nerve stimulation in cats

Splanchnic nerve stimulation in bursts at low (5 Hz) and high (50 Hz) frequency (30 V, 1 msec; train duration 1 sec; train rate 0.5/second) was employed in 10 cats under halothane anesthesia, during 10-minute periods, while blood samples were concurrently collected from the adrenal vein and femoral artery for the measurement of norepinephrine (NE), epinephrine (EPI), dopamine (DA), Met-enkephalin (ME), neuropeptide Y (NPY), peptide YY (PYY) and neurotensin (NT). In Group I (n = 5), splanchnic nerve stimulation was initially applied at 5 Hz followed after 20 min by a 50 Hz stimulus, while in Group II (n = 5) the stimulation sequence was reversed. Adrenal vein and femoral artery plasma levels of catecholamines and neuropeptides were not significantly affected by the stimulation sequence, while a significant decrease in blood pressure response was observed in Group II during the 5 Hz stimulation as compared to Group I, indicating desensitization. Splanchnic nerve stimulation at 5 Hz caused a preferential increase in adrenal vein NE (9-fold) versus EPI (7-fold) levels as compared to baseline, while 50 Hz stimulation led to further comparable increases in NE (5-fold) and EPI (6-fold) levels. Significant increases in adrenal vein DA and neuropeptide levels were only observed during 50 Hz stimulation, with DA showing a 5-fold, ME a 2.6-fold and NPY a 3-fold increase as compared to 5 Hz stimulation, and NT a 3.6-fold increase as compared to baseline. Present findings indicate different dynamics in the movement of catecholamines and neuropeptides from the adrenal.     

Identiflcation of a Proenkephalin Precursor in Striatal Tissue

Recent studies have supported the suggestion that proenkephalin is the same in both adrenal medulla and brain. However, although previous investigations have characterized enkephalin-containing adrenal intermediates derived from proenkephalin, as yet no such intermediates have been isolated from the brain. This has led to the belief that the processing of proenkephalin in the brain is extremely rapid and enkephalin-containing intermediates do not accumulate. In this investigation Sephacryl-300 gel filtration chromatography of guinea pig striata, extracted in 8 M urea, demonstrated several peaks of both bioactive and immunoreactive enkephalin-like peptides after enzymatic digest (trypsin followed by carboxypeptidase B). Comparable profiles were obtained using rat and bovine striatal tissue. In guinea pig the major species emerging from gel filtration, eluting with an apparent molecular weight of 29,000, represented approximately 9% of the total (methionine) enkephalin immunoreactivity. It had an apparent pI of 5.0 when subjected to chromatofocusing. This species was further characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and nitrocellulose blotting techniques as well as highly specific radioimmunoassays to (Met5)-enkephalin, (Leu5)-enkephalin, and (Met5)-enkephalin-Arg6-Phe7. This species was found to contain these opioid peptides in an approximately 6:1:1 ratio, respectively, and to have an apparent molecular weight of 31,000. It was also indicated that (Met5)-enkephalin-Arg6-Phe7 constituted the C-terminal seven residues of this molecule.     

Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Increased amidation

We have used antisera directed towards eight different portions of the proenkephalin molecule to examine the processing rates and patterns of proenkephalin-derived peptides in chromaffin cell cultures in the presence and absence of reserpine. Reserpine treatment produced profound effects on the molecular weight profile of nearly all enkephalin-containing peptides. Increased production of low molecular weight immunoreactive [Met5]enkephalin, [Leu5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, and [Met5]enkephalin-Arg6-Phe7 was observed in reserpine-treated cultures; immunoreactivity corresponding to several intermediate sized enkephalin-containing peptides such as Peptide B and the high molecular weight [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive peptide was decreased. The production of two amidated opioid peptides, amidorphin and metorphamide, was greatly accelerated in the presence of reserpine. The increased levels of low molecular weight enkephalins could not be accounted for by assuming decreased basal release. These results indicate that reserpine treatment is able to increase the extent of post-translational processing of proenkephalin within chromaffin cells.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Differential Release of Enkephalin and Enkephalin-Containing Peptides from Perfused Cat Adrenal Glands

We have compared the enkephalin-like material derived from proenkephalin released from perfused cat adrenal glands stimulated with pilocarpine (5 X 10(-4)M) and nicotine (5 X 10(-6) M). In addition, two doses of acetylcholine (10(-5) and 10(-4) M) and 50 mM K+ were tested. Free Met-enkephalin immunoreactivity and total Met-enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin-containing fragments, were coreleased with catecholamines. Free Met-enkephalin immunoreactivity represented 13% of total immunoreactivity for nicotinic stimulation, 46% for pilocarpine, 33% for 10(-5) M acetylcholine, 22% for 10(-4) M acetylcholine, and 16% for 50 mM K+. Analysis of the perfusate by gel filtration showed that 80% of the total Met-enkephalin immunoreactivity whose release was induced by pilocarpine was eluted in fractions corresponding to fragments of low molecular weight, whereas these fractions accounted only for 10% of the total Met-enkephalin immunoreactivity whose release was induced by nicotine. HPLC analysis of low-molecular-weight peptide fractions revealed that Met-enkephalin, Met-enkephalin-Arg-Gly-Leu, and Met-enkephalin-Arg-Phe represented 69% of total Met-enkephalin immunoreactivity whose release was induced by pilocarpine. These results indicate that selective activation of muscarinic receptors is followed by release of low-molecular-weight material, whereas nicotine application also yielded high-molecular-weight peptides. Furthermore, increasing the acetylcholine concentration from 10(-5) to 10(-4) M and using 50 mM K+ increased proportionally the high-molecular-weight peptide secretion. Results are discussed in relation to the existence of a heterogeneous population of granules either in the same cell or in different cells, containing proenkephalin-derived peptides. (ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of opiate agonists on growth hormone and prolactin release in rats

Various opioid receptor agonists, including Met5-enkephalin amide, Leu5-enkephalin amide, [D-Ala]2-Met5-enkephalin amide, [D-Ala]2-Leu5-enkephalin amide, morphine sulfate, d-methadone hydrochloride, and l-methadone hydrochloride were administered to adult male rats by subcutaneous injection. All opioid receptor agonists except Leu5-enkephalin amide significantly stimulated growth hormone and prolactin release. Naloxone and naltrexone blocked the hormone stimulatory effects of the opioids and both naloxone and naltrexone, when administered alone, significantly reduced serum growth hormone and prolactin concentrations. The dopaminergic agonist apomorphine, but not the alpha-adrenergic agonist clonidine, blocked opiate stimulation of prolactin. Morphine sulfate caused growth hormone release in rats pretreated with alpha-methyl-p-tryosine, a catecholamine synthesis inhibitor. Cholinergic agonists, physostigmine and pilocarpine, antagonized the growth hormone and prolactin release induced by morphine sulfate. The data suggest that the opiates stimulate prolactin via an interaction with catecholaminergic neurons controlling prolactin release and stimulate growth hormone via a mechanism independent of alpha-adrenergic or general catecholaminergic influence. The mechanism through which cholinergic agonists act to inhibit opiate agonist stimulation of growth hormone is presently unknown.     

The release of catecholamines from the adrenal medulla and its modulation by alpha 2-adrenoceptors in the anaesthetized dog

The adrenal nerve of anaesthetized and vagotomized dogs was electrically stimulated (10 V pulses of 2 ms duration for 10 min) at frequencies of 1, 3, 10, and 25 Hz. There was a correlation between the frequency of stimulation and the plasma concentrations of epinephrine, norepinephrine, and dopamine in the adrenal vein, mainly after the 1st min of stimulation and the maximal concentration was reached sooner with higher frequencies of stimulation. Moreover, the relative percentage of catecholamines released in response to the electrical stimulation was not changed by the frequency of stimulation. To test the hypothesis that a local negative feedback mechanism mediated by alpha 2-adrenoceptors exists in the adrenal medulla, the effects of the systemic administration of clonidine (alpha 2-antagonist) on the concentrations of catecholamines in the adrenal vein were evaluated during the electrical stimulation of the adrenal nerve (5 V pulses of 2 ms duration for 3 min) at 3 Hz. Moreover, the effects of the systemic injections of more specific alpha 2-agonist and antagonist (oxymetazoline and idazoxan) were tested on the release of catecholamines in the adrenal vein in response to electrical stimulation of the splanchnic nerve at 1 and 3 Hz frequencies. The injection of 0.5 mg/kg of yohimbine caused a significant increase in the concentrations of epinephrine and norepinephrine in the adrenal vein induced by the electrical stimulation of the adrenal nerve and the injection of 15 micrograms/kg of clonidine had no effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Appearance of enkephalin-immunoreactivity in rat adrenal medulla following treatment with nicotinic antagonists or reserpine

Various neuroendocrine factors known to be important in the regulation of adrenal catecholamine biosynthesis were investigated for possible effects on enkephalin-like immunoreactivity (Enk-IR) in the adrenal medulla of the rat. In normal rats, the adrenal chromaffin cells were not stained for either methionine (met-) or leucine (leu-) Enk-IR. Staining for Enk-IR appeared in many chromaffin cells following denervation of the adrenal or treatment of rats with the nicotinic receptor antagonists chlorisondamine or pempidine. These observations suggest that splanchnic nerve activity normally depresses the levels of enkephalin-like peptides in chromaffin cells through a trans-synaptic mechanism involving acetylcholine release and nicotinic receptor stimulation. Paradoxically, treatment with reserpine also increased Enk-IR in chromaffin cells. However, this increase did not appear to result from the well known effect of reserpine to increase presynaptic nerve firing and tyrosine hydroxylase (TOH) activity, since no increase in Enk-IR was observed following treatment with phenoxybenzamine or 6-hydroxydopamine, drugs which also increase TOH activity through trans-synaptic mechanisms. The reserpine effect also did not appear to be mediated by a stress-induced increase in glucocorticoid hormones since glucocorticoid therapy alone did not increase adrenal Enk-IR. It is suggested that the increase in adrenal Enk-IR following reserpine may result from a direct action of reserpine on chromaffin cells.     

Nicotinic receptor stimulation enhances enkephalin-like peptides processing in chromaffin cells

Acute stimulation of chromaffin cells in cultures with acetylcholine (ACh), 1,1-dimethyl-4-phenylpiperazinium (DMPP), or high potassium gave rise to a significant increase in the release of [Met5]-enkephalin immunoreactive material (ME-IRM) into the assay medium. The cellular content of ME-IRM following the actual release induced by these secretagogues remained constant suggesting the replenishment of the cellular peptides. The repletion of the peptides may occur through an enhancement of the processing rate of the proenkephalin precursor. Furthermore, the increase in secretion as well as the repletion of the cellular ME-IRM were calcium-dependent and were inhibited by the nicotinic receptor antagonist, hexamethonium, but not by atropine. These results indicate that secretion and repletion of the peptides are tightly coupled and activated by nicotinic receptor stimulation.     

Effects of Opioid Peptides Containing the Sequence of Met5-Enkephalin or Leu5-Enkephalin on Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.     

Met5-enkephalin-arg6-phe7 and its receptor in lung

The presence of met5-enkephalin-arg6-phe7 (YGGFMRF) and opiate receptors in rat, guinea pig and human lung was investigated with specific and sensitive radio immuno- and radio-receptor assays. 1) High and low molecular weight YGGFMRF-like immunoreactivity were detected in lung extracts using Bio-Gel P-2 column chromatography followed by radioimmunoassay. Using HPLC, we determined that the low molecular weight YGGFMRF-like immunoreactivity is authentic YGGFMRF. 2) The contents of YGGFMRF were 0.68 +/- 0.08, 0.76 +/- 0.12 and 0.63 pmol/mg protein in lung of rat, guinea pig and human, respectively. In the lung of these three species, the content of YGGFMRF is much greater than that of met5-enkephalin. 3) 47 mM KCl released YGGFMRF from rat lung slices in a Ca++ dependent manner. 4) Rat lung membranes were shown to bind [3H]-etorphine in a saturable manner. There are two populations of binding sites with a Kd = 0.6 and 7.1 nM and a Bmax = 7.8 and 28.5 fmol/mg protein, respectively. This binding could be displaced by YGGFMRF with high affinity, the other endogenous opioid peptides were poor displacers. From these results, we infer that YGGFMRF might be a putative neurotransmitter or neuromodulator, its role in the regulation of lung function can now be investigated.     

Met5-enkephalin-arg6-phe7 content of human and rabbit plasma

Using an adsorption technique combined with high pressure liquid chromatography and a specific radioimmunoassay, the met5-enkephalin-arg6-phe7 (YGGFMRF) content was measured in human and rabbit plasma. This heptapeptide content was 0.16 +/- 0.03, 0.20 +/- 0.05 pmol/10 ml plasma, for human and rabbit plasma, respectively. The degradation of YGGFMRF injected intravenously (rabbit) or that of the opioid heptapeptide added to rabbit plasma is rapid. The biological half life (T 1/2) of 125I-YGGFMRF in rabbit plasma was about 45 seconds (in vivo) and 1 minute and 8 seconds (in vitro). The metabolic clearance rate of YGGFMRF is slower than that of met5-enkephalin. The YGGFMRF content of rabbit plasma increased following inhibition of dipeptidyl peptidase activity by an intravenous injection of captopril. The presence of met5-enkephalin-arg6-phe7 into circulation indicates that it may have some physiological role, however it is unknown whether circulating YGGFMRF originates from adrenal medulla, intermediate lobe of pituitary or some other site.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers.

In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu5]-enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid- and phenylpropionic acid-based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C-terminal amino acids of opioid peptides (L-Leu, [Leu5]-enkephalin; D-Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N-terminal amino acid (L-Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30-50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes-Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu5]-enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.