Hypothalamic Peptidases Research Articles

The role of EC 3.4.24.15 in the post-secretory regulation of peptide signals

In the present studies, we characterized the degration of gonadotropin-releasing hormone (GnRH) by tissues of the ovine hypothalamo-pituitary axis. Membrane and soluble fractions of the medial basal hypothalamus, the pre-optic area, the median eminence and the anterior pituitart demonstrated greater GnRH-degrading activity than either hypophysial-portal or jugular plasma. The primary stable product of the membrane fractions was GnRH 1–3, while the major product of the soluble fractions was GnRH 1–5; both fragments were generated by plasma. Of all tissue fractions, the highest specific activity was observed in the soluble median eminence. Partial purification and characterization of soluble hypothalamic peptidase activity suggested that GnRH degradation by this tissue occurs via a two-step mechanism involving both post-proline cleaving enzyme and the metalloendopeptidase 3.4.24.15.

Endogenous opioid peptides and hypothalamic neuroendocrine neurones.

This consideration of the influence of endogenous opioid peptide systems on GnRH and oxytocin neurones serves to illustrate some of their possible regulatory interactions with other neuroendocrine systems. Opioids are known to influence the secretion of all the anterior pituitary hormones (see Grossman & Rees, 1983) and these effects are likely to be mediated, at least in part, in the hypothalamus. For example, inhibitory effects of opioids have also been described on secretion from the median eminence of somatostatin (Drouva et al. 1981b) and dopamine (Wilkes & Yen, 1980), and this site of action probably accounts for at least some of the stimulatory effects of exogenous opioids on plasma growth hormone and prolactin levels respectively. For the GnRH neurones the influence of endogenous opioid neurones, possibly the arcuate beta-endorphin system, appears to be mediated indirectly by inhibiting release of excitatory or facilitatory monoamines. This opioid-adrenergic interaction itself appears to be central in the regulation of gonadotrophin secretion and mediation of the feedback effects of gonadal steroids in the brain. The steroids may act directly on both adrenergic and opioid neurones, altering monoamine metabolism and release which may, in turn, regulate numbers of adrenergic receptors perhaps located on the GnRH neurones. Opioid peptide levels are also modulated by steroids probably reflecting altered synthesis and/or processing of precursors. Regulation of the opioid-adrenergic input may not only acutely affect the secretory output of the GnRH neurones but also influence synthesis or processing of GnRH itself (see Kalra & Kalra, 1984) and its degradation by hypothalamic peptidases (Advis, Krause & McKelvy, 1983). Oxytocin neurones demonstrate three further levels of interaction with endogenous opioid peptides. First the anatomical organization of the oxytocin neurones has enabled a clear demonstration of the action of opioids close to the secretory terminals to uncouple the generation of electrical activity from release of peptide. Secondly, both the oxytocin and the neighbouring vasopressin neurones themselves synthesize, process and package opioid peptides. These neurones thus provide a clear example of co-existence of several biologically active products in individual neurones. The relative expression of the different gene products may prove to be a further level of control of opioid influences on the oxytocin and vasopressin neurones.(ABSTRACT TRUNCATED AT 400 WORDS)

36] Measurement of the degradation of luteinizing hormone releasing hormone by hypothalamic tissue

This chapter describes the strategies and presents the methodologies, used to assess the enzymic degradation of luteinizing hormone releasing hormone (LHRH) by discrete regions of the rat hypothalamus. The chapter reveals that to assess the physiological relevance of peptidase activity, it is important to examine enzymic activity in relation to the activity of LHRH secretory neurons. This chapter describes the approach to the assessment of hypothalamic peptidase activity acting on luteinizing hormone releasing hormone (LHRH). The chapter also discusses its possible physiological relevance as related to the activity of the LHRH hypophysiotropic neuronal system that regulates gonadotropin secretion. The chapter describes the strategies that is adaptable for the study of peptidase activities acting on a variety of neuroendocrine peptides in discrete hypothalamic regions, or, indeed, for the investigation of neuropeptidase activity at any site in the nervous system.

Neurotensin release by rat hypothalamic fragments in vitro

The release of neurotensin by hypothalami from male rats was investigated in vitro using tissue fragments incubated in Krebs-Ringer bicarbonate-glucose buffer at 37 degrees C. Neurotensin was measured by radioimmunoassay using an antibody directed toward the C-terminal portion of the peptide. Neurotensin-like immunoreactivity released into the incubation medium eluted from Sephadex G-25 in a position identical to that of the synthetic peptide and serial dilutions of incubation medium were parallel to those of synthetic neurotensin in the radioimmunoassay. Neurotensin-like immunoreactivity released into the incubation medium was degraded during the incubation period by released hypothalamic peptidases. The addition of bacitracin (0.5 mg/ml) to the medium partially prevented this degradation. Neurotensin release was stimulated by dibutyryl cyclic AMP (10(-4) M) and by depolarizing concentrations of potassium. The latter effect was shown to be Ca2+-dependent. Dopamine (10(-4)--10(-6) M) stimulated neurotensin release in a dose-dependent manner and this effect was blocked by the dopamine receptor antagonist, haloperidol. Neurotensin release was not stimulated by either norepinephrine (10(-4) M) or serotonin (10(-4) M). The results indicate that neurotensin is released by the hypothalamus in vitro; its release is stimulated by membrane depolarization in a Ca2+-dependent manner and may involve an adenylate cyclase mechanism; and dopamine appears to serve as a stimulatory neurotransmitter for neurotensin-containing neurons.

Inactivation of Two Hyperactive LH-RH Analogues by Rat Hypothalamic Peptidases

Hyperactive analogues of luteinizing hormone-releasing hormone (LH-RH) are beleived to derive their properties from either increased binding affinity to anterior pituitary receptor sites or through decreased susceptibility to enzymic degradation. To investigate the latter suggestion and to examine the possible sites of hypothalamic peptidases inactivating LH-RH, D-Ser(TBU)6-EA10-LH-RH and D-Leu6-EA10-LH-RH, which are known to have considerably greater activity than LH-RH, were incubated with a hypothalamic supernatant fraction containing active peptidases degrading LH-RH, and their gonadotrophin-releasing ability after incubation with the enzymes was tested in normal, adult male rats; LH-RH was also tested in the same way. From a comparison of the relative losses of biological activity, both the LH-RH analogues treated proved to be more resistant to the hypothalamic peptidases than LH-RH itself; the D-Leu6-EA10-LH-RH retained its gonadotrophin-releasing activity longer than the D-Ser(TUB)6-EA10-LH-RH. These findings indicate that increased activity of the analogues may, in part be due to increased resistance to enzymic inactivation and suggest initial sites of cleavage at the Gly-leu and Pro-Gly NH2 bonds in the LH-RH decapeptide by the hypothalamic enzymes. Studies on the action of peptidases on LH-RH and its analogues may yield useful information in the design of peptidase with increased biological activity.

Enzymatic degradation of somatostatin by rat plasma and hypothalamus.

A highly sensitive and specific radioimmunoassay for somatostatin has been used to study inactivation of the neurohormone by plasma and hypothalamic peptidase(s). Specificity of the inactivation process was indicated by the absence of interference by addition of luteinizing hormone releasing hormone, thyrotropin-releasing hormone, oxytocin, or substance P. The inactivating ability of hypothalamic tissue and plasma was destroyed by heating and the protease inhibitor benzamidine prevented plasma activity, thus suggesting the enzymatic nature of the processes involved. The present data suggest that the inactivation of somatostatin by hypothalamus and plasma could be an important factor in the regulation of circulating somatostatin levels.

Enzymic inactivation of neurotensin by hypothalamic and brain extracts of the rat

A sensitive and specific radioimmunoassay has been developed to study inactivation of neurotensin by hypothalamic and brain peptidases. Degrading activity of peptidases from both hypothalamus and brain seems to have similar activity. These peptidases are temperature- and time- dependent. Brain and hypothalamic enzymes of particulate fractions can be differentiated on the basis of the pH effects; brain peptidase(s) has (have) maximal activity at pH 7.4 and hypothalamic peptidase(s) displaying a maximal activity at pH 5.8. Kidneys and liver extracts contain enzyme(s) degrading neurotensin.

Luteinizing hormone-releasing hormone (LH-RH) production and degradation by rat medial basal hypothalami in vitro

The in vitro production of hypothalamic LH-RH was examined by measuring the differences in tissue content of this neurohormone in incubated versus non-incubated rat medial basal hypothalami. A highly significant increase in hypothalamic LH-RH content was noted which was proportional to the duration of incubation. This observed increase was completely inhibited by incubating tissues at 0 °C, and was not found to occur in similar sized pieces of lateral cortex. A much smaller production of LH-RH was seen by incubation of the preoptic hypothalamic area. In all experiments very little LH-RH was detected in the incubation media. In experiments comparing physiologically different animals, LH-RH production was greater in adult males than in prepubertal males or females, and was higher in intact adult males than in castrates of the same age. Enzymic LH-RH degradation was investigated by adding exogenous synthetic hormone to either the incubating hypothalami or various tissue homogenates and measuring its disappearance as a function of time and/or concentration. These experiments demonstrate a hypothalamic peptidase(s) which degrades LH-RH at a V max of 1.7 pg/min/μg of protein (24 °C) with a K m of 2.1 × 10 −8 M/1. Peptidase activity was found in several areas of the brain as well as in the pituitary. Furthermore, there was considerable enzymic activity present in the media from the incubated hypothalami rendering it infeasible to accurately quantitate LH-RH secretion.

Higher molecular weight immunoreactive species of luteinizing hormone releasing hormone: Possible precursors of the hormone

Separation of extracts of sheep hypothalami on Sephadex G-25 gave three peaks exhibiting luteinizing hormone releasing hormone immunoreactivity. One peak corresponded in elution volume with luteinizing hormone releasing hormone but the others (I and II) eluted earlier, indicating that they are of higher molecular weight. Elution volumes were unaffected by 8 M urea treatment. Incubation of I and II with hypothalamic peptidases produced a small quantity of immunoreactive material eluting in the luteinizing hormone releasing hormone region. Digestion of I with trypsin resulted in a marked increase in total immunoreactivity and the production of material with the same elution volume as II. Tryptic digestion of II gave rise to a small quantity of immunoreactive peptide eluting in the luteinizing hormone releasing hormone region. The amount of I and II relative to luteinizing hormone releasing hormone was lower in the median eminence than in the supra optic chiasmatic and basal hypothalamic regions.

The effects of gonadectomy and gonadal steroids on the activity of hypothalamic peptidases inactivating luteinizing hormone-releasing hormone (LH-RH)

The effects of gonadectomy and gonadal steroids on the activity of hypothalamic peptidases inactivating luteinizing hormone-releasing hormone (LH-RH)

Competitive inhibition between oxytocin and luteinizing hormone-releasing factor (LRF) for the same enzyme system in the rat hypothalamus.

ABSTRACT From the observation that hypothalamic peptidases inactivating oxytocin might be involved in luteinizing hormone-releasing factor (LRF) metabolism and that the hypothalamus contains enzymes which can inactivate LRF, competitive inhibition studies were performed to determine if the peptidases inactivating oxytocin could act on LRF. In mixed substrate incubations, LRF significantly decreased peptidase activity in a hypothalamic supernatant fraction whereas thyrotrophin-releasing factor (TRF) had no effect. From kinetic studies, it was found that there was competitive inhibition between the two polypeptides for the same enzymes and that LRF may be true enzyme substrate. The results confirm the previous proposals that the hypothalamic peptidases can inactivate LRF and the possibility of a role for the enzymes in the regulation of reproductive function is suggested.

The development of an in vitro system for estimating oxytocinase in the rabbit hypothalamus and the effects of some protein synthesis inhibitors and oestradiol-17 beta on enzyme activity.

ABSTRACT An in vitro system for investigating the effects of steroid hormones and protein synthesis inhibitors on hypothalamic peptidases inactivating oxytocin has been developed. In the presence of oestradiol-17β enzyme activity was increased in the in vitro system whilst this increase was blocked completely by cycloheximide and partially blocked by actinomycin-D. It is apparent therefore that oestradiol-17β acts directly on the hypothalamus stimulating oxytocinase activity.

Changes in hypothalamic peptidase activity during the oestrous cycle in the adult female rat.

ABSTRACT The activity of peptidases in the rat hypothalamus which are capable of inactivating oxytocin has previously been found to vary with stimuli known to influence gonadotrophin release and may be related to both luteinizing hormone (LH) and luteinizing hormone releasing factor (LH-RF) release (Griffith & Hooper 1972a,b). In the present study, enzyme activity was determined in normal female rats during the morning and afternoon of each stage of the oestrous cycle, in normal rats, and in female rats injected neonatally with testosterone. The activity of the supernatant fraction was found to be not significantly different during the morning of each stage, but was greatly decreased on the afternoon of pro-oestrus; particulate activity did not vary during the oestrous cycle. Supernatant and particulate activities were found to be the same in normal male rats and testosterone-treated females, as previously shown. Both fractions' activities were significantly less than those found in the oestrous cycle, other than on the afternoon of pro-oestrus. These results indicate changes in hypothalamic peptidase activity during the oestrous cycle which may be inversely related to LH and LH-RF release; they also confirm the masculinizing effect of neonatal testosterone on the hypothalamus.

Peptidase activity in the hypothalami of rats treated neonatally with oestrogen.

ABSTRACT The previous paper (Griffiths & Hooper 1973) described the activity of certain hypothalamic peptidases following orchidectomy and testosterone propionate injection, and suggested that changes in enzyme levels may be used as an index of gonadotrophin release in male rats, in a similar way to that previously described for female rats (Griffiths & Hooper 1972a). Using this approach, the effect of neonatally administered oestrogen on the hypothalamus was investigated. The marked increase in supernatant activity in male rats and the equally marked decrease in supernatant activity in female rats, both injected during the critical period of hypothalamic sexual differentiation, are interpreted as indicating decreased LH secretion in males and increased LH secretion in females respectively. It can be concluded that the changes in reproductive function produced by neonatally administered oestrogen are caused by alterations in LH-RF metabolism and that the peptidases in the rat hypothalamus are responsible for this metabolism.

The distribution of hypothalamic peptidases in pregnant and non-pregnant dogs.

It appears to be generally accepted that oxytocin and vasopressin are synthesized in the hypothalamus and stored in the neurohypophysis. A variation in the hormone content of the neurohypophysis in physiological conditions involving the release of large amounts of oxytocin or vasopressin might be expected. For example, it has been established that dehydration and lactation deplete the neurohypophysis of hormones or neurosecretory material (for references see Ortmann, 1960). Some change in the metabolism of oxytocin might be expected during pregnancy. However, the concentration of hormones in pituitaries of pregnant rats did not differ greatly from that found in non-pregnant animals (Acher, Chauvet & Olivry, 1956). In view of this observation, it was decided to find out whether or not the hypothalamic enzymes inactivating oxytocin (Hooper, 1962) varied during pregnancy, for, if this were the case, it would appear probable that the metabolism of oxytocin in the hypothalamus changed during pregnancy. The present paper describes the intracellular distribution of hypothalamic enzymes inactivating oxytocin in a series of pregnant and non-pregnant dogs, and discusses certain points arising from the observed distribution patterns.