Degradation Of LHRH Research Articles

Protein and peptide degradation in the intestine of the common brushtail possum (Trichosurus vulpecula).

A protein (bovine serum albumin: BSA) and a peptide (luteinizing hormone releasing hormone: LHRH) were used to evaluate proteolytic activity in the intestine of common brushtail possums (Marsupiala, Trichosurus vulpecula). Luminal and mucosal extracts were isolated from the duodenum, jejunum, ileum, caecum, proximal colon and distal colon, their protein content assessed and specific activities in metabolising LHRH and BSA determined in vitro. The degradation of LHRH by luminal extracts was compared with that by the pancreatic enzymes, chymotrypsin, trypsin, and elastase. The protein concentration (microg x mg-1) of mucosal extract in the duodenum was higher ( P<0.05) than in the proximal colon, but that of luminal extracts did not differ significantly between regions. Proteolytic activity of luminal extracts was greater ( P<0.01) in the jejunum and ileum than in the hindgut. In the small intestine, proteolytic activity of luminal enzymes far exceeded that of mucosal enzymes ( P<0.05). All three pancreatic enzymes hydrolysed LHRH, but chymotrypsin had the greatest activity. This study has demonstrated that, in possums, proteolysis occurs primarily in the small intestine through luminal enzymes, with chymotrypsin playing a major role. The possum hindgut contributes little to the metabolism of peptides and proteins, identifying it as a potential site to target for their absorption following oral delivery.

Activity of pancreatic endopeptidases towards luteinizing hormone-releasing hormones

LHRH and its analogues have low oral bioavailability; this is in part due to their degradation by peptidases present in the intestinal lumen. To determine the appropriate inhibitors to co-administer with LHRH oral formulations, the peptidases involved in their digestion have to be identified. Human (hLHRH) and salmon (sLHRH) LHRH analogues contain a number of potential cleavage sites for the lumenal pancreatic secreted serine endopeptidases: chymotrypsin, trypsin and elastase. The rate of LHRH degradation by equimolar concentrations of chymotrypsin, trypsin and elastase were examined separately in vitro , at pH 8.0, 15°C. At a molar ratio of 1:1000 (enzyme:LHRH), both LHRH analogues were rapidly hydrolysed by α-chymotrypsin with half-lives of 2.5±0.3 and 2.7±0.4 min (mean±S.D., n=3), respectively, whereas in the presence of elastase both LHRH analogues were slowly hydrolysed with half-lives of 90±15 and 114±21 min (mean±S.D., n=3), respectively. Trypsin had no activity towards either LHRH analogues after 2 h incubation. The degradation of the LHRH analogues by elastase is likely to be a property of the chymotrypsin impurity. It is concluded that protection of the LHRH analogues from α-chymotrypsin is a requirement for the development of oral absorbable product.

Development of a Skin Permeation cell to Simulate Clinical Study of Iontophoretic Transdermal Delivery

AbstractA new single-compartment iontophoretic skin permeation cell was developed to simulate the clinical application of iontophoretic devices. The stability and permeation kinetics of LHRH, the model peptide, were investigated in this new permeation cell (cell design A) using two analytical methods and compared with a commonly used skin permeation cell (cell design B). A polyacrylamide-based hydrogel device was fabricated as the drug reservoir for the iontophoretic transdermal delivery of LHRH. The skin permeation profiles of LHRH were found identical for both permeation cells when assayed by radioactivity measurement. However, cell design A gave a skin permeation profile that was substantially higher than that obtained in cell design B when assayed by HPLC. This is because the electrochemical degradation of LHRH occurred in the receptor compartment of the Valia-Chien permeation cell (cell design B). This degradation could be overcome by using the new single-compartment iontophoretic permeation cell.

Median Eminence and Anterior Pituitary Degradation of Luteinizing Hormone Releasing Hormone in Hens Undergoing Changes in Luteinizing Hormone Secretion

Studies described in this report provide physiological evidence for a possible involvement of median eminence (ME) and anterior pituitary (AP) luteinizing hormone releasing hormone degrading activity (LHRH-DA) in the genesis of the hen’s preovulatory surge of luteinizing hormone (LH). Serum LH and progesterone (P4), ME LHRH content and LHRH-DA, and AP LHRH-DA were determined in laying hens in the following reproductive conditions: 1) a “spontaneous” preovulatory LH surge; 2) a “premature” preovulatory LH surge; and 3) an ovulatory failure induced by feed withdrawal. The premature preovulatory surge of LH occurred 3 h after P4 administration and was preceded by an increase in both ME and AP LHRH-DA and by a decrease in ME LHRH content 1 h after P4 administration. However, the premature preovulatory LH surge was associated with a decrease in LHRH-DA back to control levels as ME LHRH content increased, even though LHRH was presumably being released from the ME at this time to maintain the preovulatory surge of LH. Although similar changes in LHRH-DA (an increase in both ME and AP LHRH-DA, followed by a decrease) also preceded the spontaneous preovulatory surge of LH, its profile was significantly blunted and no changes in ME LHRH content were associated with this LH surge. In contrast, ovulatory failure was correlated with a decrease in ME LHRH content but no changes in LHRH-DA. Therefore, in the hen, ME and AP enzymatic degradation of LHRH I might be involved in the genesis of a premature preovulatory surge of LH.

Orchidectomy Induces Temporal and Regional Changes in the Processing of the Luteinizing Hormone-Releasing Hormone Prohormone in the Rat Brain

The structures of the complementary DNA (cDNA) that encode the rat and human hypothalamic LHRH prohormones were recently determined and the corresponding amino acid sequences deduced. In addition to the LHRH decapeptide, the prohormone contains a 56-amino acid peptide sequence which has been designated gonadotropin-releasing hormone associated peptide (GAP). In the present study we examined the effect of orchidectomy on LHRH prohormone processing in three brain regions known to contain the various elements of the LHRH neuronal system; the preoptic-anterior hypothalamic area, the hypothalamus (HYP), and the median eminence. Both LHRH-like and GAP-like immunoreactivities (LHRH-LI and GAP-LI) were quantitated in these regions in intact male rats and male rats orchidectomized for 1, 2, 3, 5, 7, 14, or 40 days. In addition, the LHRH and GAP immunoreactivities were localized and the effects of orchidectomy examined using immunocytochemical techniques. One day after orchidectomy, a small decrease in GAP-LI and an increase in LHRH-LI were evident in extracts of the preopticanterior hypothalamic area which resulted in a significant decrease in the GAP/LHRH molar ratio. A similar selective decrease in GAP-LI and in the GAP/LHRH molar ratio was also observed in extracts of the HYP, but not until 2 days after orchidectomy. The GAP/LHRH ratio in both regions gradually returned to that of the intact controls. In both the HYP and median eminence, GAP-LI and LHRH-LI gradually declined in parallel through 14 days after orchidectomy. The gradual loss of immunoreactivity in these regions was evident by both RIA quantification and immunocytochemistry. These observations suggest that 1) production and processing of both LHRH and its prohormone are affected by testicular factors, 2) that divergent enzyme and/or transport systems, regulating LHRH and GAP processing, may exist at the level of the perikarya, and 3) long term orchidectomy results in decreased biosynthesis of the prohormone and/or in parallel increases in LHRH and GAP degradation or transport.

Differences between in vitro and in vivo degradation of LHRH by rat brain and other organs.

Homogenates of brain, pituitary, liver, lung, ovary, and testes were incubated with [pyro Glu1-3,4-3H]luteinizing hormone-releasing hormone ([3H]LHRH), and the profiles of metabolites generated as a function of time were determined. After 5 min of incubation, 5 was the predominant metabolite in most homogenates. Although the profiles of metabolites varied at different time intervals, metabolites 2, 3, 4, and 5, and in some instances 7 and 9, appeared to form simultaneously and were detectable at 10 min. Neither metabolite 6 nor other larger metabolites formed initially as dominant degradation products. The findings suggest cleavage of LHRH by the simultaneous action of several endopeptidases. After a single vascular transit of [3H]LHRH, metabolites were determined in the venous blood of liver, lung, and brain of rats in vivo. There were no metabolites of [3H]LHRH in venous blood of liver and lung; however, metabolites 2-4 were present in venous blood of the brain. Incubation of rat anterior pituitary cells with [3H]LHRH yielded metabolites 1-4 but not metabolites 5 or 9 as in homogenates. Incubation of [3H]LHRH with porcine follicular granulosa cells resulted in the generation of metabolites 2-7 and 9, similar to the profile in homogenates. Thus, since homogenates contain enzymes of disrupted cells, they do not always reflect mechanisms for in vivo hydrolysis of circulating LHRH. Brain degraded 12.1% of LHRH during a single vascular transit and may account for substantial degradation of the circulating hormone.

Orchidectomy induces temporal and regional changes in the synthesis and processing of the LHRH prohormone in the rat brain.

Recently, the sequence of the cDNA which encodes the LHRH-prohormone was elucidated from human placenta and human and rat hypothalamus and the corresponding amino acid sequence deduced (Seeburg et al., 1984 and Adelman et al., 1986). In addition to LHRH, the prohormone contains a 56 amino acid sequence, designated gonadotropin-releasing hormone associated peptide (GAP), which is attached to the C-terminus of the LHRH decapep-tide. Although not yet confirmed, the human GAP sequence has been reported to possess both gonadotropin-releasing and prolactin inhibiting activity (Nikolics et al., 1985). Additionally, a 13-amino acid fragment of the human GAP sequence (proLHRH 14–26) has been reported to stimulate gonadotropin release (Millar et al., 1986). Regardless of whether the non-LHRH portion of the LHRH prohormone contains biological activity, this sequence can serve as a valuable marker for studies of LHRH prohormone synthesis, processing and degradation. In order to initiate these types of studies, we have generated specific antisera (MC-1, 2 and 3) against a fragment of the human GAP sequence (proLHRH 38–66) and developed a radioimmunoassay procedure for the quantitation of GAP (Culler and Negro-Vilar, 1986). The antisera are specific for midportion sequences of the GAP molecule and do not cross-react with any other known brain peptide.

Calmodulin involvement on the Ca++-dependent release of LHRH and SRIF in vitro.

Mediobasal hypothalamic (MBH) slices of male adult rats were superfused at 37 degrees C with oxygenated Hepes-buffer Locke medium. Bacitracin (2 X 10(-5) M) was added to prevent enzymatic degradation of LHRH and SRIF. 6 min pulse of K+ (56 mM), veratridine (15 microM) or the ionophore A 23187 (10(-5) M), markedly stimulated the release of both neuropeptides. Trifluoperazine, a calmodulin inhibitor, decreased the K+-evoked LHRH and SRIF release in a dose-dependent manner; it was also effective in inhibiting the veratridine-induced neuropeptides release. Phenytoin, a calmodulin-dependent kinase inhibitor, also decreased in a dose-dependent manner the K+-induced LHRH and SRIF release; the basal release of both neuropeptides remained unaffected by either treatment. The ionophore-stimulated release of both neuropeptides was significantly inhibited as well. These data demonstrate that a Ca++-calmodulin kinase system may be involved in the mechanism of depolarization-induced LHRH and SRIF release from hypothalamic nerve terminals.

Evidence that endopeptidase-catalyzed luteinizing hormone releasing hormone cleavage contributes to the regulation of median eminence LHRH levels during positive steroid feedback.

In previous studies we provided evidence that the degradation of LHRH is regulated so as to contribute to the establishment of appropriate levels of the decapeptide during the events leading to gonadotropin secretion in the first estrous cycle at puberty in the rat. In the present report, we present the first evidence that this apparent regulation of LHRH degradation can be studied in an experimental positive feedback model. We show that LHRH degradation in the median eminence was decreased 3 h after progesterone administration, at a time when LHRH content in this region is increasing, and when serum levels of LH remained at basal levels. Six h after progesterone administration, at the time of the LH surge, median eminence LHRH degradation was still low and LHRH content had fallen to basal levels. Additionally, we exploited this model to examine the mechanism of peptidase activity change by showing that blockade of noradrenergic neurotransmission by diethyldithiocarbamate abolishes the inhibition of LHRH degradation observed prior to the secretion of LH. We conclude that the degradation of LHRH by an endopeptidase may contribute to the regulation of LHRH levels appropriate for gonadotropin release, and that this can be studied in the ovariectomized, estrogen-progesterone-treated rat.

Mechanism of degradation of LH-RH and neurotensin by synaptosomal peptidases

The products of degradation of LH-RH and neurotensin by synaptosomes isolated from rat hypothalamus and cortex have been identified. LH-RH is cleaved at Tyr 5-Gly 6 and Pro 9-Gly 10 giving rise to LH-RH (1–5), LH-RH (6–10) and LH-RH (1–9). Neurotensin is cleaved at Arg 8-Arg 9, Pro 10-Tyr 11 and Ile 12-Leu 13, giving neurotensin (1–8), neurotensin (1–10), neurotensin (1–12) and neurotensin (9–13) as major products. While most of the peptidase activity is localized in the cytoplasmic fraction, a small but significant proportion is membrane bound. For LH-RH, the specificity of the membrane-bound activity is similar to that in the cytosol fraction; for neurotensin, the membrane fraction preferentially gives rise to the (1–10) and (1–11) peptides. The most potent inhibitors of the LH-RH and neurotensin degrading enzymes in synaptosomes are heavy metal ions (mercury and copper), p-chloromercuribenzoate and 1,10 phenanthroline.

Luteinizing hormone-releasing hormone peptidase activities in discrete hypothalamic regions and anterior pituitary of the rat: apparent regulation during the prepubertal period and first estrous cycle at puberty.

Presently, two peptidase activities degrading LHRH have been observed in rat hypothalamic tissue: an endopeptidase and postproline cleaving enzyme (PPCE). Since no clear evidence for their physiological role in the onset of puberty has been presented, we examined the relationship between the content and degradation of LHRH by the median eminence (ME) and that by the ventromedial preoptic area (POA) during the prepubertal period and during the first estrous cycle at puberty. LHRH degradation by anterior pituitary during this period was also determined. Using kinetically optimized conditions, total LHRH degradation was measured by high performance liquid chromatographic estimation of the loss of synthetic LHRH catalyzed by low speed tissue supernatants, and PPCE activity was measured using a fluorogenic substrate (carbobenzoxy-1-glycl-1-prolyl-4- methylcourmarine-7-amide). The ME and POA LHRH content and serum levels of LH were estimated by RIA. During the prepubertal period, in animals killed between 22 and 30 days of age, the total LHRH-degrading activity in the ME remained constant (50 pmol LHRH degraded/μg protein- 30 min), while the LHRH content increased steadily. During the pubertal period (first estrous cycle), a high correlation between LHRH degradation and LHRH content in the ME was observed (r = 0.95). In animals killed between 34 and 38 days of age, total LHRH degradation increased from basal levels in anestrous animals to higher levels in early proestrous rats, reaching the highest value on the morning of late proestrous (300% increase). After the morning of late proestrus, LHRH degradation decreased (by 1300 h; P < 0.01) to reach prepubertal values on the first estrus, the following day. On diestrous day 1, both total LHRH degradation and LHRH content returned to early pubertal values. Cleavage at Pro9-Gly10 of LHRH (PPCE) was unchanged throughout the period studied. Both total LHRH degradation and postproline degrading activity on LHRH showed only minor fluctuations during the first estrous cycle in POA and anterior pituitary samples. The data suggest that LHRH degradation may contribute to the regulation of LHRH levels appropriate for gonadotropin release during the onset of puberty.

Mechanism of luteinizing hormone-releasing hormone degradation by subcellular fractions of rat hypothalamus and pituitary.

The pathway of LH-RH degradation by two subcellular fractions (a soluble fraction and a 25 000 X g particulate fraction) of rat hypothalamus, pituitary and cerebral cortex has been studied using high performance liquid chromatography and amino acid analysis to identify the breakdown products. The primary cleavage point in the Tyr5-Gly6 bond giving [1-5] LH-RH and [6-10] LH-RH. In the presence of dithiothreitol, cleavage of LH-RH also occurred at the Pro9-Gly10 bond giving [1-9] LH-RH. The fragment [1-5] LH-RH is further degraded sequentially from the C-terminus and [1-4] LH-RH, [1-3] LH-RH, tyrosine and tryptophan were identified. The other major fragment, [6-10] LH-RH, is rapidly broken down, the only intermediate product positively identified being Arg-Pro.

Dopamine stimulates the degradation of gonadotropin releasing hormone by rat synaptosomes.

Immunohistochemical studies have shown the existence of synapses between nerve endings containing dopamine and gonadotropin releasing hormone (LH-RH) respectively in the hypothalamic median eminence. It is generally accepted that this transmitter is involved in the regulation of gonadotropin secretion as a modulator of LH-RH release, although the evidence is conflicting and both inhibitory and excitatory actions of dopamine have been reported. In vitro studies support the excitatory effect and low doses of dopamine have been shown to release LH-RH from incubated hypothalamic fragments and synaptosomes. This report describes studies with nerve endings isolated from the rat hypothalamus in which dopamine at higher concentrations than those used previously has been found to stimulate the degradation of LH-RH but not thyrotropin releasing hormone (TRH). This mechanism seems to be physiological in that it is calcium-dependent, requires the structural integrity of the nerve endings and fluctuates with the reproductive state of the animal.

Resistance to enzymic degradation of LH-RH analogues possessing increased biological activity

Three analogues of LH-RH in which Dextrarotatory amino acids were substituted for the Gly 6, and two additional analogues in which the Leu 7 residue was also modified, were subjected to enzymic preparations derived from rat hypothalamus or anterior pituitary. These enzymes, known to cleave LH-RH, preferentially at the Gly 6-Leu 7 position, proved less effective in degrading all the analogues tested. Among the Gly 6 substituted analogues, [D-Trp 6] LH-RH, having the highest LH-releasing activity, was most resistant to degradation. Additional modification, at position 7, although rendering the analogues immune to enzymic attack, did not further enhance their biological potency. These data suggest that degradation of LH-RH is a physiological determinant of its biological activity and has therefore to be considered with on designing new, potent analogues of the hormone.

In vitro release of luteinizing hormone-releasing hormone (LHRH) from rat mediobasal hypothalamus: effects of potassium, calcium and dopamine.

A suitable preparation to study the effect of dopamine (DA) on in vitro LHRH secretion is presented. Enzymatic degradation of LHRH in the incubation medium is completely inhibited by bacitracin (2 X 10(-5) M). Ahigh potassium concentration (56mM) induces an increase in LHRH release from entire pieces of mediobasal hypothalamus (MBH), but not from a synaptosomal pellet; this release is inhibited when calcium is omitted. The depolarization-induced LHRH in the MBH. In contrast, DA (10(-6)M) is not effective in vitro on MBH from ovariectomized rats but induces a fast release of LHRH from MBH of normal male and estradiol-pretreated ovariectomized rats. The preparation presented here appears to be of great interest in investigating amine-steroid-LHRH interactions at the cellular level.

LHRH‐PITUITARY PLASMA MEMBRANE BINDING: THE PRESENCE OF SPECIFIC BINDING SITES IN OTHER TISSUES

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.