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Peptide and Protein Hormones----4. Corticoliberin – Proopiomelanocortin Cascade

Author:N/A    | Post time:2012-05-26

4. Corticoliberin – Proopiomelanocortin Cascade
Top of page
1. Introduction
2. Gonadoliberin, Thyroliberin, Gonadotropins, Thyrotropin, Inhibin, and Related Hormones
3. Parathyroid Hormone and the Calcitonin Family
4. Corticoliberin – Proopiomelanocortin Cascade
5. Blood Pressure Regulating Peptides
6. Cholecystokinin and Gastrin
7. Secretin Family
8. Neurotensin [1971]
9. Motilin [2005]
10. Pancreatic Spasmolytic Peptide
Corticoliberin (corticotropin releasing hormone or factor, CRH or CRF) and the peptides derived from proopiomelanocortin (POMC) are called the stress hormones because they are released in response to stress. The hypothalamic factor, CRH, stimulates cAMP synthesis and a cAMP-dependent protein kinase [431], resulting in secretion of POMC from the corticotrophic cells of the adenohypophysis, the endocrine cells of the pars intermedia, and central neurons that originate in the hypothalamus. The prohormone, POMC (h-POMC has 241 amino acids, Mr 26 678) is then enzymatically cleaved to form POMC-(1 – 108)-NH2 (HP-N-108), corticotropin (ACTH), and -lipotropin (-LPH), see Figure 3. Further degradation of HP-N-108 gives POMC-(1 – 76) and HP-N-30 [POMC-(79 – 108)-amide] [432]. The former is degraded to POMC-(1 – 28) [433], -melanotropin (-MSH), and -MSH [434]. ACTH can be cleaved to form -MSH and the corticotropin-like intermediate lobe peptide (CLIP). Cleavage of the N-terminal tetrapeptide of CLIP gives -cell tropin (-CTP), and -LPH yields -LPH, -MSH, -endorphin (-EP), and the melanotropin potentiating factor (MPF). Acetylation by opiomelanotropin acetyltransferase increases the biological activity of -MSH, decreases the activity of ACTH, and abolishes the opiate activity of -EP.  Figure 3. Degradation of proopiomelanocortin (POMC)
 


In the adrenal cortex, ACTH stimulates the formation of the glucocorticoids and -MSH releases aldosterone in the presence of angiotensin II (AT II). The gluco- and mineralocorticoids play an important role in stress situations. The glucocorticoids enhance liver gluconeogenesis and exhibit immunosuppressive and anti-inflammatory activity. For further details, see  Hormones – Adrenal Steroid Hormones . The release of ACTH and -EP is inhibited by the glucocorticoids via a negative feedback mechanism. The mineralocorticoid aldosterone exerts antidiuretic and antinatriuretic effects which are important during blood loss.

Both CRH and POMC-derived stress hormones usually inhibit release of the reproductive hormones [435] and enhance release of the atrial natriuretic factor (ANF). ANF then down regulates the stress hormones and stimulates release of the reproductive hormones; it is presumably important as a regulator in coping with stress. The androgens and gonadotropins stimulate POMC synthesis [436], whereas estrogen and testosterone inhibit the formation of the stress hormones [437]. Expression of the POMC gene in the testes correlates closely with the maturation of the Leydig cells. In contrast to the adenohypophysis, the POMC gene in the testes is not regulated by glucocorticoids [436].

Permanent stress probably desensitizes the immunosuppressive ACTH – glucocorticoid system. This condition is characterized by chronic fatigue and increased formation of antibodies against the body\'s own tissues, a preliminary stage of autoimmune diseases [438].

The structures of h- [439], r- [440], b- [441], sa-, and xe-POMC [442] have been elucidated via their DNA sequences.

  4.1. Corticoliberin
  Occurrence. Corticoliberin [9015-71-8], also known as corticotropin releasing hormone or factor (CRH or CRF), is found in nerve fibers throughout the brain, in the pancreas [443], adrenal medulla [444], placenta [445], stomach, and testes [446].

Although h-CRH is identical to r-CRH (peptide amide with 41 amino acids, Mr 4757), the precursor proteins of these two species have different sequences [447]. p-CRH [448] is very similar to h- and r-CRH.

  Release. There is a circadian rhythm in the secretion of CRH. Similar to ACTH, the plasma level of CRH in humans peaks at 6 a.m. and has its lowest values at 6 and 10 p.m. [449]. CRH is released in response to stress (e.g., hunger [450], loss of blood [444], peripheral increase in noradrenaline [451]). Very small doses of catecholamines stimulate and large doses inhibit the central release of CRH [452].

The pulse frequency of CRH in the rat is greatly increased by the chronic administration of alcohol [453]. The release of CRH from the hypothalamus is also stimulated by morphine [454], acetylcholine [455], and interleukins [456]. Endogenous glucocorticoids appear to be important for the synthesis and release of CRH.

The release of CRH from the hypothalamus is inhibited by ACTH [457] and endogenous opioids [458] via a negative feedback mechanism. Very small doses (0.1 – 1 nmol/L) of -endorphin stimulate the release of CRH in vitro, but higher doses (> 0.1 µmol/L) inhibit both basal and acetylcholine-stimulated release [459].

CRH is involved in the paracrine formation of ACTH in the placenta. Plasma CRH levels increase continually during pregnancy from the second trimester up to birth and fall rapidly after delivery [460].

In Alzheimer\'s disease, a reduced CRH level is observed in the brain [461]. Patients suffering from anorexia nervosa [462] and depression [463] exhibit enhanced release of CRH.

Receptors. CRH receptors are found in the adenohypophysis and in regions of the brain that are related to the limbic and autonomic nervous systems [464].

In spite of the increased plasma ACTH level in adrenal-ectomized rats, the number of CRH receptors in the hypophysis falls. This down regulation is due to enhanced secretion of vasopressin [465]. In contrast, the CRH receptors on membranes of the liver, spleen, testes, prostate, and pancreas [466] increase in number after adrenalectomy.

  Biological Effects. CRH stimulates the synthesis and release of proopiomelanocortin as well as its degradation in the adenohypophysis and placenta [467] to form ACTH, -melanotropin (-MSH) [468], 3-MSH [469], and -endorphin (-EP). It also increases plasma levels of corticosterone, aldosterone [470], and 18-hydroxycorticosterone [471] via release of ACTH. CRH also directly potentiates ACTH-stimulated synthesis of corticosterone [472]. The physiological importance of CRH is emphasized by the fact that CRH antigens cause atrophy of the adrenal glands and lower plasma levels of ACTH and -EP.

Extracellular Ca2+, the entry of Ca2+ into the cell, calmodulin [473], and cAMP-dependent protein kinase [474] all play a role in the CRH-stimulated release of ACTH.

Intravenously applied CRH raises the plasma level of oxytocin and vasopressin, which potentiate the activity of CRH [475]. Angiotensin II [476] and gastrin-releasing peptide-(14 – 27) [477] also potentiate the ACTH-releasing effect of CRH. Somatostatin, melanophore concentrating hormone [478], delta-sleep-inducing peptide [479], glucocorticoids, morphine [480], and endogenous opiates inhibit the ACTH-secreting effect of CRH.

In vitro, CRH stimulates the release of endorphin and dynorphin from the rat pituitary [481]. Intravenous dynorphin A-(1 – 13) and intravenous or intradermal CRH inhibit the inflammatory response to scalding in rats independent of the pituitary and adrenal functions [482]. The anti-inflammatory effect is not dependent on the hypophysis, but on adrenal function [483].

CRH inhibits the reproductive functions of both sexes [484], [485]. The release of GnRH to the hypophysis from the hypothalamus is prevented by CRH [486]. The prolactin-dependent inhibition of GnRH release is partly due to the activation of CRH-containing neurons [487]. CRH also centrally inhibits the release of growth hormone by stimulating somatostatin release in the hypothalamus [488]. Intravenous CRH raises plasma levels of prolactin [489].

High intravenous doses of CRH selectively dilate the blood vessels of the mesenterium; this is responsible for a decline in peripheral resistance and systemic arterial blood pressure. CRH activates prostaglandin synthesis. The prostaglandins, in turn, inhibit the hypotensive effect of CRH by a negative feedback mechanism [490]. The central application of CRH raises blood pressure and pulse rate by increasing adrenaline and noradrenaline levels. In vivo, CRH potentiates the cholecystokinin-induced pancreatic secretion of protein and the secretin-induced secretion of  [491].

Intravenous CRH results in a strong and persistent lowering of the level of pentagastrin-stimulated gastric acid, while the plasma gastrin level increases [492]. Intraperitoneal CRH prevents formation of stress-induced gastric ulcers in rats [493]. CRH inhibits the emptying of the stomach [494], reduces the transit in the small intestine, increases the transit in the large intestine, and causes diarrhea [495].

In humans, intravenous CRH increases the breath-time volume, breath frequency [496], and attentiveness; it also improves the mood and well-being of depressive patients [497].

CRH exerts a central antipyretic effect [498]. In experimental animals (mainly rats) central application of CRH increases locomotoric activity, emotionalism [499], and cleaning behavior; reduces food uptake [500] and aggressiveness; and enhances defensive behavior [501].

Structure – Activity Relationships. The three amino acids at the N-terminus of CRH are not important for biological activity. However, vasodilating and ACTH-releasing activity fall to below 0.1 % on further deletion of N-terminal amino acids.

The C-terminal region is of extreme importance for CRH activity. Both 41-deamido-o-CRH-(1 – 41) with a free C-terminal carboxyl group and o-CRH-(1 – 39)amide have < 0.1 % of the activity of CRH. Met21 is of some significance for activity because [Met(O)21]o-CRH exhibits only 10 % of the biological activity. However, norleucine and norvaline can be substituted for this amino acid with practically no loss of activity.

  Uses. Subcutaneous injection is advisable and is equally as effective as intravenous administration. Activity after intranasal administration is only about 1 % of that obtained on parenteral administration [502].

A pulsatile injection of CRH normalizes ACTH and cortisol secretion after glucocorticoid therapy (secondary adrenal insufficiency) [503] and apparently does not desensitize the pituitary [504]. Pulsatile application (6.25 µg every 30 min) is preferable to long-term infusion (50 or 100 µg/4 h) [505].

CRH can be used as a diagnostic aid for hypophyseal function [506]. After intravenous administration of 100 µg of CRH, the plasma ACTH and cortisol levels in patients suffering from Cushing\'s syndrome increase to a higher extent than in healthy persons [507]. In depressive patients with a high level of cortisol, CRH produces a lower ACTH response than in normal persons [508]. An abnormal increase in growth hormone after application of CRH is observed in patients suffering from congenital thyrotropin deficiency [509].

  4.2. Corticotropin [510]
  Occurrence. Corticotropin [9002-60-2], also known as adrenocorticotropic hormone (ACTH), has 39 amino acids, Mr 4541 (h-ACTH), and is formed in the adenohypophysis from the precursor POMC. It is also found in the interstitial fluid of the testes [511] and in the pancreas [512]. Enzymatic degradation and acetylation of ACTH give rise to the N-terminal derivative -melanotropin (-MSH) and two C-terminal derivatives: the corticotropin-like intermediate lobe peptide (CLIP) and -celltropin (-CTP). ACTH sequences from various species are given in [513].

  Release. ACTH is formed from the precursor POMC in response to stress, CRH, and stimuli that release CRH or potentiate its activity. The formation of ACTH is accompanied by an increase in cAMP (see Section Corticoliberin) [510]. ACTH is secreted in micropulses lasting a few minutes. Pulse frequency remains the same throughout the day, but the amplitude is higher in the morning than in the afternoon and night [514], it is also higher in men than in women [515]. Serotonin increases the formation of ACTH and cortisol by stimulating release of CRH. It appears to be partly responsible for the circadian increase in cortisol [516].

In vivo, corticosteroids inhibit the secretion of ACTH [510] in the adenohypophysis and cerebrospinal fluid. Glucocorticoids do not affect the in vitro CRH-stimulated release of ACTH from the pituitary [517]. -Aminobutyric acid (GABA) appears to reduce the release of ACTH via inhibition of CRH release [516]. Increased salt intake depresses the formation of ACTH in response to stress or CRH [518].

Inhibitors of angiotensin converting enzyme inhibit the release of ACTH via decreased synthesis of angiotensin II.

ACTH secretion is increased in Cushing\'s syndrome, but lowered in Sheehan\'s syndrome. The circadian ACTH rhythm is disturbed in patients suffering from depression; hypersecretion of ACTH and cortisol is observed [519].

Receptors. An ACTH receptor (Mr 225 000) from mouse adrenal cells consists of four different subunits [520]. In humans, ACTH receptors have also been found on splenocytes and peripheral mononuclear leukocytes [521].

IGF-I potentiates the ACTH-stimulated formation of ACTH receptors [522] and TGF- reduces the number of ACTH receptors on adrenocortical cells [523].

  Biological Effects. In the adrenal cortex, ACTH stimulates the synthesis of glucocorticoids (cortisol, cortisone, corticosterone) and mineralocorticoids (cortexone and aldosterone) in a calcium-dependent process [524] that involves activation of adenylate cyclase. ACTH must be administered in the morning for maximum stimulation of glucocorticoid synthesis. The sensitivity of adrenal cells to ACTH is reduced when they are exposed to ACTH for longer periods [525].

The ACTH-stimulated secretion of aldosterone probably proceeds via angiotensin II [526]. ACTH also enhances the release of adrenaline and noradrenaline in the adrenal gland [527]. It exerts a trophic effect on the adrenocorticotropic cells of the adrenal gland. Adrenal hyperplasia is observed as a result of the hypersecretion of ACTH (Cushing\'s disease). The adrenal glands shrink as a result of ACTH insufficiency of the pituitary (Sheehan\'s syndrome).

Corticostatin from granulocytes is a peptide containing 34 amino acids that inhibits the ACTH-stimulated formation of corticosterone [528].

ACTH and N-terminal fragments of ACTH, e.g., ACTH-(4 – 10) [529] also have a therapeutic effect in cases of shock [530]. Cholinergic mechanisms in the central nervous system play an important role here [531].

ACTH-(1 – 24) stimulates the pancreatic secretion of NaHCO3 and protein via cholinergic mechanisms [532]. The ACTH molecule also has insulinotropic and hypoglycemic activity [512]. However, it also potentiates the diabetogenic activity of growth hormone induced by hypoglycemia [533].

ACTH binds specifically to splenocytes, inhibiting the production of antibodies and interferon (direct immunosuppressive activity) [521].

ACTH-(4 – 10) is responsible for the influence of ACTH on behavior and on learning processes (see Section Melanotropins): it increases attentiveness, sexual activity, and the memory capacity of rats, at the same time reducing anxiety. Cholinergic neurons and muscarinic receptors play a role in ACTH-dependent behavior patterns [534].

The central application of ACTH, ACTH-(1 – 24), and -MSH decreases food uptake [535]. Centrally applied ACTH increases blood pressure and heart rate, it also prevents persons from falling asleep, while de-Ac--MSH and CLIP intensify slow-wave sleep and paradoxical sleep [536].

Structure – Activity Relationships. A fragment as small as ACTH-(4 – 7) (10–4 mol/L) stimulates in vitro corticosterone synthesis. A dramatic increase in activity occurs with the inclusion of Met3. However, the full activity of the N-terminal sequence is attained with a free amino group at the N-terminal Ser1. Thus, ACTH-(1 – 10) is 100 times more active than ACTH-(5 – 10), and ACTH-(1 – 18) has the full activity of native ACTH.

ACTH-(1 – 24) (tetracosactid) is more active in releasing corticosteroids than ACTH in vitro. ACTH can be divided into at least four segments. Sequence (11 – 18) is very important for receptor binding. Sequence (4 – 10) is responsible for the corticotropic effect, and the N-terminal tripeptide (1 – 3) is the “amplifier”. The C-terminal fragment, ACTH-(25 – 39), is responsible for antigenicity and safe transport. ACTH-(11 – 24) is a competitive antagonist for the corticotropic activity of ACTH. [Phe9]ACTH-(1 – 24) also inhibits glycolysis and steroidogenesis.

ACTH from guinea pigs in which Pro24 is replaced by Ala has a higher aldosterone-releasing activity than hACTH or ACTH-(1 – 24)amide [513]. [Cys(carboxamidomethyl)25]ACTH-(1 – 26) has three to four times the activity of ACTH in stimulating aldosterone secretion, but a lower corticosterone-stimulating activity. The substitution of d-Ser or -Ala for Ser1, of Lys for Arg17, and of Lysin-amide or 1,4-diaminobutylamine for Arg18 gives long-acting analogues that have about up to eight times the activity of ACTH.

[-Ala1, Lys17]ACTH-(1 – 17)-4-amino-n-butylamide (ACTH-(1 – 17), alsactide) is stable to aminopeptidases and carboxypeptidases and has a long duration of action.

CLIP exhibits insulin-releasing activity which decreases after shortening or acetylation of the molecule at the N-terminus [537]. CLIP, like -MSH and de-Ac--MSH, lowers the -endorphin-stimulated secretion of prolactin in rats [538].

  Uses. Natural ACTH from the pig is marketed under the name Acethropan (Hoechst). The synthetic ACTH derivatives, tetracosactide (Synacthen, CIBA) and alsactide (Synchrodyn 1 – 17, Hoechst), are also used. ACTH preparations are used as diagnostic aids for the functioning of the adrenal cortex and as a therapeutic agent for insufficient functioning of the adrenal cortex, in multiple sclerosis, inflammatory rheumatic diseases, collagen diseases, acute gout, radicular pain syndrome, severe allergic skin diseases, and collitis ulcerosa. ACTH also has an antiemetic effect and is administered to cancer patients treated with cis-platinum [539].

  4.3. Melanotropins
  Occurrence. -Melanotropin [37213-49-3], also known as -melanocytes stimulating hormone (-MSH), Mr 1665, is derived from POMC (POMC-112 – 124). In mammals its amino acid sequence is

Ac–S Y S M E H F R W G K P Va

It is found primarily in the hypophyseal pars intermedia and the hypothalamus.

- and -MSH are derived from POMC-(1 – 28) and -MSH from -LPH (see Fig. 3). In many species, two different -MSH peptides are found.

  Release. Release of -MSH is probably regulated through the enzymatic degradation of oxytocin as the prohormone for melanostatin (MIF) and melanoliberin (MRF). Stress and corticoliberin increase brain and plasma -MSH levels. -Endorphin can also release -MSH in the central nervous system. The -MSH is released in the septum in response to fever induced by interleukin-1, especially when the temperature increases (shivering phase) [540].

Increased levels of -MSH are observed in physiological stress, cardiovascular distress, blood loss [541], and cardiac arrest [542].

  Biological Effects. Melanin is a black-brown skin pigment that consists mainly of polymerized dihydroxyphenylalanine. In melanocytes, -MSH stimulates tyrosinase and thus the synthesis of melanin (melanogenesis); it also promotes pigment transport of pigment granules (melanosomes). Dispersion of the melanosomes through the numerous dendrites of the melanocytes causes the skin to darken, the skin is lightened by aggregation of melanosomes. Human skin becomes darker within 24 h after administration of -MSH. Darkening of the skin is observed in kidney damage and diseases in which the plasma -MSH and ACTH levels are increased (Cushing\'s, Addison\'s, Nelson\'s disease, and ACTH-secreting tumors). The skin becomes lighter in hypophyseal insufficiency. The C-terminal tetrapeptide of -endorphin, the melanotropin potentiating factor (MPF), enhances the melanotropic activity of -MSH.

The second messenger of -MSH appears to be cAMP. Inhibitors of -MSH (e.g., melatonin) inhibit the -MSH-stimulated formation of cAMP but increase the concentration of cGMP. Melatonin, a hormone found in the pineal body, stimulates the aggregation of melanosomes.

-MSH has many other activities in mammals [543]:

Increase in testicular Sertoli cell cAMP

Secretion of estradiol and plasminogen activator

Increase in lipogenesis and sebum production in the skin

Increase in lipolysis in adipose tissue

Increase in plasma free fatty acid levels

Increase in adrenal steroidogenesis related to fetal growth and development

Increase in aldosterone synthesis and secretion by the adrenal zona glomerulosa (angiotensin II seems to be important [544])

Increase in pineal serotonin levels

Decrease in pineal melatonin levels

Increase in growth hormone secretion from the pituitary

Inhibits stress and -endorphin-stimulated prolactin release [545]

Increase in plasma luteinizing hormone [546]

Increase in plasma glucagon and insulin

Decrease in blood pressure

Decrease in plasma calcium levels

Decrease in bone resorption

Decrease in immunomodulatory and inflammatory activities of IL-1 [547]

Reduction of body temperature following intracerebroventricular or parenteral administration [548]

Behavior (increased arousal, attention, learning, memory retention, sexuality)

Improved nerve regeneration


h--MSH has the same activity as -MSH in the Anolis skin test (darkening of the skin). -MSH sequences from other species also show high melanotropic activity, which is usually slightly lower than that of -MSH. Like -MSH, -MSH also raises the plasma levels of glucose, glucagon, insulin, and free fatty acids in the rabbit.

Of the -MSH peptides, Ac-1-MSH exerts the highest melanotropic effect but still has < 0.1 % of the -MSH activity. 2-MSH inhibits the release of -MSH from the hypophysis and reduces the -endorphin-induced analgesic and hypothermic effect. The direct infusion of -MSH into the renal artery results in prompt excretion of sodium and potassium [549]. The ACTH-stimulated synthesis of glucocorticoids and aldosterone is potentiated by 3-MSH [550]. Intraventricular application of -MSH leads to a longterm increase in blood pressure [551]. The functions of POMC-(1 – 108) and its fragments are probably based on its growth-promoting effect on the adrenal cortex and its hypertensive effect.

Structure – Activity Relationships. The N-terminal acetyl group is important for the melanotropic activity of -MSH. -MSH can be subdivided into three regions:

The classical messenger sequence, His-Phe-Arg-Trp (-MSH-(6 – 9))

The C-terminal tetrapeptide Gly-Lys-Pro-Val-amide which has seven times the melanotropic activity of -MSH-(6 – 9)

The N-terminal sequence Ac-Ser-Tyr-Ser-Met-Glu which acts as a potentiator

Within the messenger sequence, -Phe-Arg- appears to be crucial for melanotropic activity. The minimum effective sequence is Ac-His-Phe-Arg-Trp-amide [552]. The lysine in the C-terminal tetrapeptide is important for activity. Met4, Gly10, and Pro12 are important for MSH activity [552]. If Met4 and Gly10 are replaced by a cysteine disulfide bridge ([Cys4,Cys10]-MSH), a cyclopeptide is obtained which contains the two important messenger sequences. This peptide is a superagonist and is 10 000 times more active than -MSH in the frog skin test. The cyclic analogue Ac-[Cys4,Cys10]-MSH-(4 – 10)amide is less active than -MSH, whereas Ac-[Cys4,Cys10]-MSH-(4 – 13)-amide is again superactive and has the same activity as [Cys4,Cys10]-MSH in the frog skin test. Thus, the C-terminal sequence is of great significance for MSH activity. The disulfide bridge is important for biological activity. Reduction of the disulfide bridge results in a 1000 – 10 000-fold decrease in biological activity [553].

Met4 is sensitive to oxidation, and can be replaced by norleucine without loss of activity. Treatment of -MSH with hot alkali prolongs the duration of action. [Nle4,d-Phe7]--MSH is 60 times as active as -MSH in the frog skin test. In mice, [Nle4,d-Phe7](4 – 11) or -(4 – 10)-MSH has 100 times the activity of -MSH [554]. Even Ac-[Nle4,d-Phe7]-MSH-(4 – 9)amide is still ten times as active as -MSH in the melanoma tyrosinase and lizard skin tests, but it is ten times less active in the frog skin test [555]. The central application of [Nle4,d-Phe7]-MSH has an antipyretic effect that is ten times greater than that of -MSH. The antipyretic effect after intravenous application is, however, not pronounced. The C-terminal tripeptide appears to be very important for antipyretic activity [556].

Cyclic compounds containing d-Phe7, an acidic amino acid in position 5, and a basic amino acid in position 10 are more active than -MSH [557].

The activity of [Cys4,d-Phe7,Cys10]-MSH on melanocytes is about as high as that of [Cys4,Cys10]-MSH, but it acts considerably longer. [Cys4,d-Phe7,Cys10]-MSH-(1 – 12)amide has the same potency as [Cys4,d-Phe7, Cys10]-MSH, indicating that Val13 is not required for melanotropic activity [558].

An analogue of -MSH-(4 – 9), Met(O2)-Glu-His-Phe-d-Lys-Phe-OH (ORG-2766) [559], [560] is about 100 –1000 times more active than -MSH-(4 – 9) in promoting learning. However, high doses have the opposite effect. The subcutaneously or orally applied peptide has an antiamnesic effect, antagonizes pentobarbital anesthesia, and reduces morphine uptake in the brain. Met(O2)-Glu-His-Phe-d-Lys-Phe-NH-(CH2)8-NH2 (HOE 427, Ebiratide [561]) and Met(O)-Glu-His-Phe-d-Lys-Phe-NH-(CH2)8-NH2 have 100 times the ORG-2766 activity in this test. In learning tests with rats, HOE 427 is about 500 times more active than ORG-2766 [562]; the sequence Phe-d-Lys-Phe appears to be especially important [563].

Antagonists [543]. Ac--MSH-(7 – 10)amide is a weak, selective -MSH antagonist in the lizard skin bioassay. Ac-[d-Trp7,d-Phe10]-MSH-(7 – 10)amide is a competitive inhibitor of -MSH in the frog and lizard skin assays [564]. A strong antagonist for frog skins is the uncyclized peptide Ac-Nle-Asp-Trp-d-Phe-Nle-Trp-Lys-amide; the cyclic lactam of this compound is a full agonist. Other antagonists are the growth hormone releasing peptide, His-d-Trp-Ala-Trp-d-Phe-Lys-amide and its analogues.

  Uses. [Nle4,d-Phe7]-MSH (intermedin alpha) can be applied topically and is absorbed through the skin causing increased pigmentation in the yellow mouse [565]. It is being tested as a suntanning agent.

-MSH-(6 – 9) exerted a positive transdermal effect (better mood, as well as less anxiety, pain, spasticity, and muscular weakness) in patients suffering from multiple sclerosis [566].

ORG-2766 improved the mood and level of performance of patients without influencing their sleep [554]. Even in elderly, mentally weak patients, ORG-2766 increased attentiveness and induced social behavior [567]. Ebiratide is being tested on patients suffering from Alzheimer\'s disease.

  4.4. Opioid Peptides
Many peptides that have an effect on opioid receptors are formed from prohormones. -Endorphin (-EP, Section -Endorphin) is derived from proopiomelanocortin (Fig. 3), its activity is based on the N-terminal pentapeptide sequence Tyr-Gly-Gly-Phe-Met. This sequence is also found in Met-enkephalin (Met-EK), which is produced from prepro-EK A of the adrenal medulla. A series of extended Met-EKs that are structurally related to prepro-EK A have been isolated from the adrenal medulla (Section Peptides Derived from Preproenkephalin A). The extended Leu-EKs isolated from the pituitary and hypothalamus (e.g., dynorphins, neoendorphins, and rimorphin) are derived from a common precursor, prepro-EK B (Section Peptides Derived from Preproenkephalin B). Prepro-EK A and B are structurally similar.

The C-terminal tetrapeptide sequence of prepro-EK A is, apart from the missing amide group, identical to that of femarfarmamide (FMRF-amide), a peptide isolated from mollusks. FMRF-amide is not an opiate. It is not derived from prepro-EK A because the C-terminal glycine important for the formation of the amide group is missing. In mollusks, it is formed from a prepropeptide which contains 21 copies of the precursor FMRFG sequence.

Kyotorpin, a Tyr-Arg dipeptide with analgesic activity, was isolated from the bovine brain. It is probably derived from the propeptide neokyotorpin (Thr-Ser-Lys-Tyr-Arg).

The dermorphins and deltorphins (Section Dermorphin and Deltorphins) are heptapeptide amides from the skin of the South American frogs Phyllomedusa sauvagii, P. rhodei, and P. bicolor. They contain d-alanine or d-methionine.

The exorphins (Section Exorphins) are opioid peptides which are formed during food digestion. Examples are - and -casomorphins.

Modified morphines (e.g., naloxone or certain EK analogues) are antagonists for endogenous opioids and exogenous opiates. In a certain dosage range, cholecystokinin (CCK-8) also acts as an endogenous selective antagonist for the analgesic activity of opioids.

  Opioid Receptors. Prior to the discovery of endogenous opioids, three opioid receptors were postulated: the -receptor (for morphine), -receptor (for ketocyclazocin), and -receptor (for SKF 10 047). The discovery of the enkephalins (EK) led to the introduction of the -receptors. -EP prompted the postulation of the -receptors, and the naloxone-sensitive effect on thermoregulation gave rise to -receptors. Iota-receptors have been postulated in the dog and rabbit intestine, and -receptors with a high affinity for 4,5-epoxymorphinane have been described. The -receptors have been subdivided into 1- and 2-receptors. There are three types of -receptor [568].

All endogenous opioids are ligands for the -, -, -, and -receptors. Met-EK, Leu-EK, and -EP are the main ligands for the - and - receptors. The dermorphins are specific for the -receptors, the deltorphins for the -receptors, and the dynorphins for the -receptors. -EP binds primarily to the -receptors. Opiates (e.g., morphine) bind to the 2-receptors.

In vitro bioassays for the receptors are described in [569]. Selective ligands for the - and -receptors are described in [570]. Peptide ligands for the - and -receptors are given in [571][572][573][574].

  Biological Activity. The most important effects mediated by the opioid receptors follow [575]:

1-mediated: supraspinal analgesia, inhibition of gastrointestinal transit and motility [576], suppression of experimentally induced diarrhoea [576], and development of anorexia.

-mediated: spinal analgesia, inhibition of acetylcholine release from rat corpus striatum [577], impairment of avoidance learning in rats [578], and inhibition of SP-stimulated plasma extravasation and vasodilation [579].

-mediated: analgesia, increased food intake [580], ACTH release in rats [581], and diuresis (mediated by the adrenal medulla) [582].

-mediated: supraspinal analgesia.

-mediated: psychomimetic effects and behavioral changes.

 
4.4.1. -Endorphin
  Occurrence [583]. -Endorphin (-EP) [60617-12-1], Mr 3465 (h--EP), contains 31 amino acids and is derived from proopiomelanocortin (see Fig. 3). It has been isolated primarily from the pituitary, but is also found in human placenta cell cultures, ovaries [584], sperm [585], endometrium [586], gallbladder [587], pancreas, and small intestine.

-EP derivatives such as -EP-(1 – 27) (C fragment or -EP), Ac--EP, Ac--EP-(1 – 27) [588], and -EP-(1 – 18) are found in the rat adenohypophysis. Acetyl--EP-(1 – 18) mainly occurs in the rat neurohypophysis [589]. In rats, the degree of acetylation of the hypothalamic endorphins increases with age [590].

The C-terminal tetrapeptide of -EP (Lys-Lys-Gly-Glu in humans) is the melanotropin potentiating factor (MPF).

  Release. -EP is secreted in the hypophysis in response to stress, corticoliberin, angiotensin II, lipoxygenase or epoxygenase products [591], insulin-induced hypoglycemia (via cholinergic mechanisms [592]), adrenaline, food uptake, and chronic alcohol consumption [593]. -EP is released from the mucosa of the small intestine by gastric acid or bile acid and from the gallbladder mucosa by CCK-8 [588]. Estrogen and testosterone increase the plasma level of -EP [594]. Both h-CG and PMSG stimulate formation of -EP in the ovaries [595] and in the testicular interstitial fluid [596].

In humans, somatostatin and oxytocin have no effect on the basal plasma -EP level, but lower the increased levels of -EP, -LPH, and cortisol caused by insulin-induced hypoglycemia [597].

In humans, release of -EP exhibits a circadian rhythm similar to that of cortisol: a high level in the morning and a low level at night [598]. The basal -EP level is lower in people who bear a high risk of becoming alcoholics compared to those who do not. The plasma level of -EP increases with alcohol consumption in the high risk group, but not in the low risk group [599]. Plasma levels of -EP are significantly raised in depressive patients [600] and in obesity [601].

  Biological Effects. -EP has analgesic and lipolytic activity [602]. The intracerebroventricular application of CCK-8 [603] and -MSH inhibits the analgesic activity of -EP. -EP lowers phosphodiesterase activity, somatostatin secretion in the isolated pancreas of the dog, the release of GnRH in the mesencephal central grey substance, plasma LH, the oxytocin release induced by sucking, and, in high doses, the formation of CRH, ACTH, LPH, and cortisol. Very low doses release CRH in rat hypothalami [604].

Intraventricular and intravenous application of -EP causes release of growth hormone and prolactin. The -EP-stimulated release of prolactin can be inhibited by -MSH or CLIP [605]. -EP increases plasma insulin, glucagon, and glucose, especially in obese humans [606].

Pharmacological doses of -EP reduce the left ventricular systolic and diastolic pressure in the isolated rat heart [607]. Depending on the dose and conditions, the intracerebroventricular application of -EP results in hyper- or hypothermia. Other central effects are the release of catecholamines, Met-EK [608], thyrotropin, vasopressin, and -MSH as well as increased food uptake.

Structure – Activity Relationships. -EP has a highly specific opiate recognition sequence at the N-terminus (positions 1 – 5) which is linked to an amphiphilic helix (positions 13 – 31) by a hydrophilic region (positions 6 – 12) [609]. Since the N-terminal Tyr-Gly bond of -EP is more resistant to aminopeptidases than that of the EKs, -EP has a longer duration of action. Acetylated sa--EPs have no activity in an opiate receptor assay.

Incorporation of Gln or Arg at position 8 doubles the analgesic effect, Trp in position 27 quadruples the analgesic effect. [Gln8,Trp27]h--EP has almost eight times the receptor binding ability of h--EP, but its analgesic effect corresponds to that of h--EP. The replacement of Glu8 appears to be of great importance for receptor binding and may find application for the design of -EP antagonists [610]. [Gln8,Gly31]h--EP-Gly-Gly-amide is a strong antagonist for -EP-induced analgesia, it is 200 times more active than naloxone [611].

The substitutions d-Ala2 and MePhe4 increase the binding of the -EPs to the -receptors. Increasing hydrophobicity at position 5 correlates with decreasing analgesic activity [612].

Substitution of the dermorphin sequence for the seven amino acids at the N-terminus gives a highly analgesic peptide which is 4.4 times more active than h--EP and about as active as dermorphin [612].

Analogues which do not show homology with -EP in the twelve amino acids at the C-terminus but have the helical structure of the C-terminus are as active or more active than -EP. Cysteine bridges between positions 14 and 26, 15 and 26, 16 and 26, as well as 17 and 26 are tolerated. These derivatives exhibit stronger receptor binding than h--EP [613].

-EP-(1 – 27) has only 0.2 % of the -EP activity in the opiate receptor displacement assay. -EP-(1 – 27) inhibits -EP-induced analgesia and release of growth hormone [614], and the -EP-induced hypothermia in the mouse [615], but not the -EP-induced release of prolactin [614].

The N-terminal tyrosine is very important for the analgesic activity of -EP: de-Tyr1--EP does not bind to opiate receptors. h--EP-(6 – 31) inhibits -EP-induced analgesia, the -EP stimulated release of prolactin, but has no effect on the release of TSH [616]. h--EP-(6 – 31), h--EP-(28 – 31), and h--EP-(30 – 31) inhibits the -MSH-induced grooming, stretching, and yawning syndrome, as well as -EP-induced grooming and catatonia [617]. The C-terminus is of prime importance for the lipolytic activity of -EP. Derivatives in which the two C-terminal amino acids are deleted have no lipolytic activity [618].

 
4.4.2. Preproenkephalins A and B
There is considerable structural similarity between the two preproenkephalins (prepro-EKs) isolated from endocrine and nerve tissue. The structures of h-, b-, r- [619], xe- [620] prepro-EK A and h- and b-prepro-EK B have been elucidated. Prepro-EK A is mainly cleaved to give Met-EK and extended Met-EK peptides (e.g., peptide E and adrenorphin). Prepro-EK B is cleaved to give Leu-EK, the neoendorphins, and the dynorphins.

Prepro-EKs are widely distributed in the body and are processed differently according to their location [621].

 
4.4.2.1. Peptides Derived from Preproenkephalin A
  Occurrence. Preproenkephalin A (prepro-EK A) [88895-24-3], Mr 30 781 (h-prepro-EK A), contains 267 amino acids. Numerous peptides derived from prepro-EK A have been isolated from the adrenal medulla [622] ; these peptides are in turn precursors of Met-EK (Tyr-Gly-Gly-Phe-Met, Mr 574) and Leu-EK (Tyr-Gly-Gly-Phe-Leu, Mr 556). Plasma proteins treated with pepsin also generate peptides related to Met-EK [623].

  Release. The release of the enkephalins is stimulated by GABAnergic mechanisms, insulin-induced hypoglycemia [624], endotoxin shock [625], electroshock [626], intraventricular application of -endorphin [627], and glucocorticoids [628]. Dopamine inhibits the formation of Met-EK [629]. Synthesis of messenger RNA for prepro-EK A is stimulated by stress [630], angiotensin II [631], FSH, and cAMP [632], but inhibited by chronic administration of glucocorticoids [630]. The plasma level of Met-EK increases considerably in marathon runners [633].

Met- and Leu-EK are rapidly degraded by aminopeptidases and enkephalinase, which cleaves the C-terminal dipeptide.

  Biological Effects [634]. For a description of the activity of the enkephalins, see Section Opioid Peptides (1-, -, and -mediated opioid actions). Enkephalins inhibit transmitter release from nerve terminals in the central and peripheral nervous systems by blocking calcium channels [635].

Met- and Leu-EK mainly bind to -receptors and only have an analgesic effect if applied centrally. Analogues that preferentially bind to the - and -receptors exhibit an analgesic effect, even after peripheral application. The tachyphylaxis and substance dependency produced by morphine are also observed after chronic application of the highly active EK analogues.

Endocrine and exocrine pancreatic and gastrointestinal secretion are modulated by the enkephalins [636]. Enkephalins contract the lower esophagus and the pyloric sphincter [637], delay gastrointestinal transit [638], trigger gallbladder contraction [639], relax the Oddi sphincter [640], cause vasoconstriction in the lungs [641], and inhibit bladder motility [642].

Depending on the dosage and species, enkephalins exert a hypotensive [643] or hypertensive effect [644]. The intracerebroventricular application of - and -agonists to rats inhibits the release of oxytocin [645] and vasopressin (diuresis).

The enkephalins modulate the release of ACTH and cortisol via extrahypophyseal mechanisms [646]. They potentiate the ACTH-stimulated release of corticosterone [647] and inhibit the CRH-induced increase in the plasma levels of ACTH, -EP, and cortisol [648].

The enkephalins increase the plasma level of prolactin by inhibiting dopamine release. They stimulate the release of growth hormone via the growth hormone releasing hormone.

The enkephalins inhibit ovulation and the release of lutropin by inhibiting GnRH synthesis. Endogenous opioids inhibit gonadotropin secretion; this is dependent on the gonadal steroids. The FSH-stimulated formation of progesterone is promoted by the enkephalins [649], while the secretion of testosterone from the testes of immature rats is suppressed [650]. In experimental animals, endogenous opioids inhibit the release of TRH and, thus, the formation of TSH [651].

The endogenous opioids can also act as immunostimulants [652] and immunosuppressants [653].

Structure – Activity Relationships. Tyr1 is essential for opiate activity. However, its N-terminal amino group can be methylated, guanylated, or extended by amino acids without significantly affecting the activity. The N-allyl-EKs (particularly N-allyl-Met-EK) are, like naloxone, antagonists of morphine and EK.

Gly2 can be replaced by -aminoisobutyric acid or d-amino acids to give compounds that are considerably more active and more resistant to enzymatic degradation. The more lipophilic the substitution, the stronger the binding to the -receptors and, therefore, the analgesic activity. Substitution with hydrophilic groups increases affinity for the -receptors.

Gly3 is important for the biological activity of the EKs. Replacement of Phe4 by other amino acids leads to loss of activity. However, N-methylation or substitution by AzaPhe increases analgesic activity.

The more lipophilic the amino acid in position 5, the stronger the analgesic effect after parenteral administration. For example, the intracerebroventricular application of the O-galactosyl derivative of [d-Met2,Hyp5]EK-amide produces an analgesic effect that is 50 000 times that of morphine [654]. Position 5 does not necessarily have to be occupied by an amino acid: methioninol sulfoxide, the thiolactone of homocysteine, and substituted hydrazides are also suitable.

Shortening of the chain at the C-terminus leads to the tripeptide-N-methylphenethylamides or tripeptide-2-amino-4-methylpentane amides, with a still higher analgesic activity. H-Tyr-NH(Me)-(CH2)4-CO-NH(Me)-CH2-CH2-C6H5 also shows high analgesic activity when applied intravenously [655]. An EK analogue that is cyclized via a disulfide bridge is [d-Cys2-d-Cys5]EK-amide also has a powerful analgesic effect. All of the above highly active compounds are more stable to enzymatic degradation than the rapidly degradable natural EKs.

The hydrophilic EK analogues, [d-Arg2,Phe(NO2)4, Pro5]EK-amide (BW 942C)[656] and [d-Met(O)2, Phe(NO2)4,Pro5]EK-amide (nifaltide) [657] are effective against diarrhoea.

 
4.4.2.2. Peptides Derived from Preproenkephalin B
  Occurrence. Preproenkephalin B (prepro-EK B) [88895-25-4], Mr 28 422 (h-prepro-EK B), contains 254 amino acids. The most important peptides derived from prepro-EK B are the pentapeptide Leu-enkephalin (Leu-EK), Mr 556 [658]; the longer dynorphins (DPs); and the neoendorphins (neo-EP). The DPs and neo-EPs were first isolated from the hypothalamus, hypophysis, and duodenum of the pig. Later, DPs were found in the bovine adrenal gland [659], guinea pig heart [660], and rat duodenum [661].

 

Leu-EK Y G G F L
DP-A-8 Y G G F L R R I
DP-A-9 Y G G F L R R I R
DP-A-11 Y G G F L R R I R P K
DP-A-13 Y G G F L R R I R P K L K
 

 

 

  Release. During dehydration, immunoreactive DP is elevated in the hypothalamus but lowered in the hypophysis.

In ovariectomized rats, the level of immunoreactive DP increases in the adenohypophysis, this increase is prevented by estrogen [662]. Accumulation of immunoreactive DP-8 in the hippocampus and frontal cortex of rats appears to be accompanied by reduced learning ability [663]. After traumatic injuries, immunoreactive dynorphin increases in the spinal cord of the rat. This appears to be responsible for hind limb paralysis, an attendant symptom of traumatic injuries [664]. Immunoreactive DP levels are low in the spinal cord of schizophrenic patients [665].

  Biological Effects. See Section Opioid Peptides (-receptor-mediated opioid actions). DPs potentiate the glucose- or amino-acid-stimulated release of insulin [666], but inhibit the release of somatostatin [666], TRH [667], oxytocin [668], vasopressin [669], and counteract morphine tolerance [670].

The central application of DPs in rats decreases body temperature, increases food [671] and water intake [672], suppresses motor activity [673], reduces the response to acoustic signals [673], and leads to hindlimb paralysis similar to that observed after spinal cord injury [674].

Structure – Activity Relationships. DPs are specific ligands for the -receptors. The affinity for the -receptors increases with decreasing chain length [675]. Short-chain DPs are rapidly degraded by peptidases and therefore have a considerably shorter biological activity. DP-A-8 and DP-A-9 are assumed to have a neural transmitter or modulator function at the -receptors, and the more stable DP-A-13 and DP-A-17 to have a more hormonal function. As with other opioids, the N-terminal tyrosine is essential for opiate activity.

The basic amino acids, particularly Arg6 and Arg7, are important for the biological activity of DP-A-13 [676]; substitution of Ala for Ile8 gives a more active (2 – 9 times) compound [676]. [d-Cys2,Cys5-N-MeArg7,d-Leu8]DP-(1 – 8)ethylamide is highly analgesic, it binds more strongly to the - and -receptors than to the -receptors [677].

The substitution of d-Trp in DP-A-11 gives rise to weak, nonselective antagonists [678]. N-diallyl derivatives of DP-A-11 are also opioid antagonists with weak selectivity for -receptors [679]. De-Tyr1-rimorphin inhibits morphine-induced effects [680].

  Uses. The intrathecal application of 15 µg of DP-A-13 to cancer patients produces a nociceptive effect that lasts for >4 h [681]. [d-Ala2]DP-A-6 [081733-79-1] (Dalargin) is used to treat duodenal peptic ulcers [682].

 
4.4.3. Dermorphin and Deltorphins
  Occurrence. Dermorphin (DM) [77614-16-5], Mr 803, and deltorphin (DT) [119975-64-3], Mr 955, each contain a d-amino acid:

 

Dermorphin Y a F G Y P Sa
Deltorphin Y m F H L M Da
 

 


They were isolated from the skin of the South American frogs, Phyllomedusa sauvagii, P. rhodei, and P. bicolor. One precursor of DM contains five copies of a sequence of 35 amino acids, the C-terminus of this sequence contains the DM sequence [683]. Another precursor contains four copies of DM and one copy of DT [684]. The precursors contain alanine and methionine in the l form, which are converted to the d conformation in a posttranslational reaction.

Immunoreactive DM has also been found in the brain, adrenal glands, and the gastrointestinal tract of the rat [685].

  Biological Effects. DM binds preferentially to the -receptors. When given intravenously, it has a powerful analgesic effect (10 times that of morphine).

DM inhibits the secretion of gastric acid, the emptying of the stomach, secretion from the pancreas, and intestinal motility. It increases the plasma levels of prolactin [686], growth hormone [687], thyrotropin [688], somatostatin, gastrin, glucagon [689], blood pressure, and heart rate. It inhibits the release of ACTH, -LPH, -EP [690], LH [691], secretin, and pancreatic peptide [692].

DT binds to the -receptors. When given intracerebroventricularly it improves the memory of mice [693].

Structure – Activity Relationships. The C-terminal amide group is important for the biological activity of DM [694]. In the guinea pig ileum, [Tyr7]DM has twice the activity of DM and 1.4 times the analgesic activity [695]. The substitution of Hyp for Pro6 gives a compound with the same analgesic and gastrointestinal activity, but a higher prolactin-releasing activity [694]. [Tyr(OMe)5]DM has a higher affinity for the -receptors and [Phe5]DM binds more strongly than DM to the -receptors [696].      [d-Arg2]DM has about the same analgesic activity as DM [697]. Shortening the [d-Arg2]DMs at the C-terminus enhances activity [697]. DM guanylated at the N-terminus has a higher analgesic effect and inhibits the gastrointestinal transit [698].

In the case of the tetrapeptide analogues, amides with a bulky side chain, N-terminal guanylated compounds, and the [Sar4], [d-Arg2], and [d-Met(O)2] substitutions all increase activity. Tetrapeptide analogues are described in [699][700][701][702][703][704][705][706].

The selectivity of DM and DT for the opioid receptors depends on charge effects and the hydrophobicity of the C-terminus [707]. DT-(1 – 4)amide binds almost exclusively to the -receptors. Met6, Asp7, and Leu5 are important for binding to the -receptors.

 
4.4.4. Exorphins
The term exorphins refers to peptides with opiate activity that are produced from food during digestion.

  Occurrence. -Casomorphin-7 (-CM-7) [79805-24-6], Mr 790, was isolated from casein peptone and corresponds to b- or o--casein-(60 – 66) [708]. The more active N-terminal -CM tetrapeptide amide (morphiceptin) has been synthesized and isolated from enzymatically digested milk proteins [709].

-Casomorphins were isolated from -casein after treatment with pepsin (amino acid sequences 90 – 95 and 90 – 96). An exorphin-like sequence was found between positions 43 and 49 of -gliadin [710].

 

-CM-7 R Y L G Y L E
-CM-6 R Y L G Y L
-CM-7  Y P F P G P I
-CM-5  Y P F P G
-CM-4  Y P F P
Morphiceptin  Y P F P a
 

 

 

  Biological Effects. The -CMs have only slight opiate activity. Intragastric -CMs cause the release of somatostatin. In a process that can be reversed by naloxone, digested gluten and -CM (oral) stimulate the release of insulin and glucagon in dogs after a test meal [711].

The -CMs exert an opioid effect on intestinal electrolyte transport [712]. When applied parenterally, they stimulate the postprandial release of pancreatic peptide and the amino-acid- or glucose-stimulated release of insulin [713], increase the plasma level of prolactin [714], and inhibit the release of somatostatin, thyroliberin, and thyrotropin [715].

Structure – Activity Relationships. The N-terminal tripeptide of -CM has no activity. Morphiceptin, the N-terminal tetrapeptide amide, has the same analgesic activity as Met-EK in the guinea pig ileum assay and is a specific ligand for the -receptors. The peptides, h--CM-(1 – 4) amide (valmuceptin) and h-[d-Val4]-CM-(1 – 4)amide (devalmuceptin) bind more tightly to the -opiate receptors than morphiceptin [716].

Substitution of d-Pro or d-pipecolic acid for Pro4 gives [d-Pro4]-CM-5 (deprolorphin), [d-Pro4]morphiceptin (deproceptin, Wellcome PL 017) [717], and [d-pipecolic acid4]-CM-5 (depilorphin), which are more active than morphine both in vivo and in vitro. These compounds have a high analgesic activity in the rat [718]. Substitution of d-Phe for Phe3 increases antinociceptive activity [718]. Replacement of d-Pro2 leads to loss of opioid activity, whereas [d-pipecolic acid2]-CM-5 has a higher long-lasting analgesic effect [718]. Substitution of d-Pro for Pro4 and d-Phe for Phe3 promotes binding to the -receptors.

 
4.4.5. Femarfarmamide and Related Structures
  Occurrence. The tetrapeptide femarfarmamide (FMRF-amide), Mr 599, was isolated from mollusks. It is not derived from prepro-EK A (which also contains the FMRF sequence at its C-terminus) but is coded in a gene that contains 21 copies of the FMRF sequence [719].

Immunocytochemical methods have shown that FMRF-like material occurs in the pancreas of chicken, ileum of the dog, and in brain neurons of the frog and rat. The first FMRF-like peptide from vertebrates, LPLRF-amide, was isolated from chick brain.

Other structurally related compounds are found in many animals (e.g., cockroach [720] and hawk moth [721] ).

  Biological Effects [722]. In mollusks, FMRF-amide exerts both a stimulating and an inhibitory effect on the heart. It hyperpolarizes neurons in the snail. In rats, both FMRF-amide and LPLRF-amide [723] (intravenously and centrally applied) increase arterial blood pressure.

FMRF-amide inhibits the spontaneous or acetylcholine-induced contraction of the anterior gizzard of Aplysia california and the stomach of Navanax [724]. It also inhibits colon motility in mammals [722].

FMRF-amide has no opiate activity, but acts as an opiate antagonist, e.g., [725], [726]. However, it only binds weakly to - or -receptors [727].

On intracerebroventricular application in rats, FMRF-amide exhibits amnesic activity [728] and increases the plasma level of growth hormone [729].

Structure – Activity Relationships. The C-terminal amide group and the full length of the molecule are important for the biological activity of FMRF-amide. Acetylation and benzoylation increase its contractile effect. A hydrophobic amino acid is required in position 2, while short-chain amino acids decrease activity [730].

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