Centrally mediated effects of neurohypophyseal hormones.

The neurohypophyseal hormones oxytocin and vasopressin cause a variety of biological effects in animals which are mediated by central nervous system mechanisms. Among the best studied of these effects is the modulation of both memory processes and the development of drug tolerance and dependence. Neurohypophyseal hormones have also been shown to alter various physiological parameters such as heart rate and body temperature following central administration. In addition, these peptides can profoundly alter spontaneous, unlearned behavior in several rodent species. Many of the centrally mediated effects of neurohypophyseal hormones have been shown to be elicited at sites within the brain stem and the limbic system where vasopressin and oxytocin occur in cell bodies, axons and nerve terminals, suggesting a physiological role for these peptide effects. The various central effects of neurohypophyseal hormones involve different mechanisms which can be distinguished from one another on the basis of required dose, time-course of action, and structure-activity relationships. Thus, alterations of spontaneous behavior are mediated by putative receptors closely related to vasopressin receptors in blood vessels responsible for the peripheral pressor response while the effects on memory processes are mediated by a mechanism which is not closely related to those involved in the peripheral hormonal effects of the peptides. The influence of neurohypophyseal hormones on memory and attention may be useful clinically. A potential role for these peptides in mental disorders is discussed.

Pineal ultrastructure and indole profiles spanning the summer rise in arginine vasotocin immunoactivity.

A correlative radioimmunological-biochemical-ultrastructural study of the rat pineal gland was undertaken during the summer months when pineal arginine vasotocin (AVT) immunoactivity increases up to 200-fold. RIA confirmed a rapid rise in AVT activity during mid-August regardless of the time of day sampled. Pineal indoles were separated by HPLC and measured using electrochemical detection. Serotonin (5-HT) and 5-hydroxyindoleacetic acid levels were consistently elevated in daytime samples, and there was a significant trend for increased day and nighttime levels of 5-HT from July to September. Mid-dark levels of melatonin also exhibited a significant increase over the sample period. Nighttime levels of N-acetylserotonin mirrored fluctuations in 5-HT in the preceding photoperiod. Ultrastructural components implicated in peptide/protein and/or indole biosynthesis were quantified by stereological morphometry. The greatest amounts of rough endoplasmic reticulum stacks, lipid droplets, and annulate lamellae-like bodies coincided with peak AVT activity. Dense-cored vesicles and synaptic ribbons were consistently more frequent during the dark period. The number of dense-cored vesicles and nucleolar size tended to be greatest before and after the peak in AVT immunoactivity. These observations are consistent with the hypotheses that endoplasmic reticulum and lipid are functionally related to the synthesis and/or storage of peptide/protein factors and that numerical changes in synaptic ribbons and dense-cored vesicles are more closely related to day/night differences in indole metabolism.

Partial characterization of a renin-releasing factor from plasma and hypothalamus.

Previous studies have indicated that administration of the serotonin releaser p-chloroamphetamine HCl produces a dose-dependent increase in renin secretion through a blood-borne renin-releasing factor. The present studies were designed to partially characterize this renin-releasing factor using an in vitro kidney slice method for the bioassay of renin-releasing activity. Plasma from p-chloroamphetamine-treated, nephrectomized rats was used to obtain the renin-releasing factor, which was fractionated by ultrafiltration into fractions of molecular weight ranges of 1000 to 5000, 5000 to 10,000, and 10,000 to 20,000. The molecular weight ranges of the renin-releasing factor was determined to be between 5000 and 10,000. Since previous studies have shown that lesions in the hypothalamus prevent the effect of p-chloroamphetamine on renin secretion, we tested whether a hypothalamic extract can release renin from kidney slices. Addition of extracts of boiled rat hypothalamic tissue to the kidney slices caused an increase in renin release. Addition of cerebellar extracts produced a smaller increase in renin release, whereas addition of pituitary extracts had no effect. Fractionation by ultrafiltration of bovine hypothalamic extract revealed that the fraction with a molecular weight range of 5000 to 10,000 possessed the highest renin-releasing ability. The 1000 to 5000 (molecular weight) fraction possessed a sizeable renin-releasing activity, but the 10,000 to 20,000 fraction had no renin-releasing activity. Both bovine hypothalamus fractions (molecular weights between 1000-5000 and 5000-10,000) and plasma fraction lost their renin-releasing activity after digestion with pronase, suggesting that the renin-releasing factor or factors are peptides. These results suggest that a renin-releasing factor originate in the hypothalamus.

Hypothermia induced by centrally administered vasopressin in rats. A structure-activity study.

Vasopressin and related peptides cause short-lasting hypothermia when injected into the lateral ventricle of the rat. In the present study, the structure-activity relationships for the induction of this effect were examined. For the agonist peptides studies, the structural requirements were found to be similar to those required to cause peripheral vasoconstriction ant to induce behavioral excitation in mice. However, an antagonist of the pressor and behavioral effects of vasopressin was ineffective in antagonizing the hypothermic response. Moreover, this analog and another pressor antagonist themselves caused hypothermia. Comparison with the structure-activity relationships for other effects on the central nervous system strongly suggests that the hypothermic response is unrelated to the effects of vasopressin on consolidation of memory, development of tolerance to drugs, and mechanisms of reinforcement.

Specific antagonists of the acute behavioral response to centrally-administered vasopressin in mice.

Vasopressin and related peptides cause behavioral excitation after intracerebroventricular injection in mice. This behavioral excitation is characterized by excessive scratching and grooming behavior in the unrestrained animal and enhanced escape-directed activity in stressful situations. These effects of vasopressin were found to be blocked by the administration of analogs which act as competitive antagonists of the pressor-activity of vasopressin. The potencies of these analogs in suppressing the behavioral effect paralleled the pressor antagonist potencies. The antagonists did not cause the characteristic behavioral alterations by themselves, nor did they block grooming and/or scratching behavior induced by the structurally-unrelated substances, mescaline, bombesin and substance P. It is suggested that these antagonists provide useful tools for studying the role of endogenous vasopressin in behavior.

Bradykinin metabolism in the isolated perfused rabbit heart.

BACKGROUND: Bradykinin is a potent cardioprotective hormone, the beneficial role of which in vivo appears to be limited by its rapid metabolism. Inhibitors of peptidases that degrade endogenously formed bradykinin are themselves cardioprotective, presumably by increasing local bradykinin concentrations. As bradykinin-degrading peptidases are potential therapeutic targets, it is important to identify these enzymes in different animal models of cardiac function. OBJECTIVE: To determine the mechanism of bradykinin degradation in the coronary circulation of the rabbit, using an isolated perfused heart preparation. DESIGN AND METHODS: [3H]Bradykinin (16 nmol/l) was perfused as a bolus through the isolated rabbit heart in the presence and absence of specific peptidase inhibitors. The effluent was collected and the radiolabeled metabolites of [3H]bradykinin were separated by high performance liquid chromatography, identified, and quantified. RESULTS: [3H]Bradykinin was metabolized to the extent of 62 +/- 3% in a single passage through the rabbit coronary circulation at a physiological flow rate. The metabolites were identified as [3H]bradykinin(1-5) and [3H]bradykinin(1-7),accounting for 50 +/- 4 and 12 +/- 2% of the radioactivity, respectively. Co-perfusion with the angiotensin converting enzyme inhibitor, ramiprilat, completely blocked formation of these metabolites. CONCLUSIONS: Angiotensin-converting enzyme fully accounts for the metabolism of [3H]bradykinin in the rabbit coronary circulation. This result contrasts with data obtained using rat heart, which demonstrated a prominent role for aminopeptidase P in bradykinin metabolism in this species.

Apstatin analogue inhibitors of aminopeptidase P, a bradykinin-degrading enzyme.

Membrane-bound aminopeptidase P (AP-P) participates in the degradation of bradykinin in several vascular beds. We have developed an inhibitor of AP-P called apstatin (1) (N-[(2S, 3R)-3-amino-2-hydroxy-4-phenyl-butanoyl]-L-prolyl-L-prolyl-L-al aninam ide); IC50,human = 2.9 microM. In the rat, apstatin can potentiate the vasodilatory effect of bradykinin, reduce blood pressure in an aortic-coarctation model of hypertension, and reduce cardiac damage and arrhythmias induced by ischemia/reperfusion. In this study, we have determined structure-activity relationships for apstatin analogues as well as for other chemical classes of inhibitors using AP-P isozymes from different sources. The most potent inhibitor was one in which the N-terminal residue of apstatin was replaced with a (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl residue (6, IC50,human = 0.23 microM). The (2R,3S)-analogue of 6 was equipotent with 6 while the (2S,3S)- and (2R,3R)-analogues were considerably less potent. Apstatin analogues lacking the L-alanine or having hydroxyproline in place of the proline in the second position had reduced affinity. Certain thiol-, carboxylalkyl-, and hydroxamate-containing compounds were inhibitory in the low micromolar range. Human cytosolic AP-P isozymes and Escherichia coli AP-P exhibited different inhibitor profiles than mammalian membrane-bound AP-P isozymes. The effects of the compounds on X-Pro dipeptidase (prolidase) and leucyl aminopeptidase are also presented.

Localization of rat tryptase to a subset of the connective tissue type of mast cell.

We examined the cellular distribution of rat tryptase in rat skin, lung, small intestine, and peritoneal lavage cells by immunohistochemical techniques. Tryptase purified to apparent homogeneity from rat skin was used to generate a goat polyclonal anti-rat tryptase antibody. Tryptase-containing cells were detected in lung, skin, and peritoneal lavage cells. Small intestine mucosa, on the other hand, showed few if any tryptase-positive cells. Sequential staining with Alcian blue and anti-tryptase antibody showed that tryptase is located only in mast cells. Sequential staining with safranin to identify the connective tissue type of mast cell and anti-tryptase antibody showed that tryptase resides only in this mast cell type. However, only a subpopulation of the safranin-stained mast cells contained tryptase. In lung, 53% of the mast cells stained with safranin; 94% contained tryptase. In skin, 80% stained with safranin; only 6% contained tryptase. In peritoneal cells, more than 95% of the mast cells were stained with safranin; 20% contained tryptase. In the bowel mucosa, where few cells are stained by safranin, no cells with tryptase were detected. The percentages of cells with chymase I that also contained tryptase were 80% and 84% for lung, 4% and 7% for skin, and 15% and 13% for peritoneal cells by respective simultaneous and sequential double labeling with anti-tryptase and anti-chymase I antibodies. This study suggests that the rat connective tissue type of mast cell is subdivided into two forms on the basis of the presence or absence of tryptase, whereas rat mucosal mast cells lack this enzyme. These results contrast with those in humans, in which tryptase is present in all mast cells, but are similar to mice, in which tryptase mRNA has been detected only in the connective tissue type.

Potentiation by aminopeptidase P of blood pressure response to bradykinin.

We examined whether a specific aminopeptidase P (APP) inhibitor, apstatin, increases vasodepressor responses to bradykinin in anaesthetized rats, and whether it would augment blood pressure responses further after treatment with the angiotensin-converting enzyme inhibitor (ACEi), lisinopril. Apstatin doubled the maximum blood pressure response to bradykinin. The area under the curve (AUC), which incorporates both peak blood pressure changes and duration of response, was doubled in apstatin-treated rats vs controls and in the apstatin+lisinopril group vs lisinopril alone. These data demonstrate that APP is an important kinase in vivo.

Post-proline cleaving enzyme from lamb brain.

A post-proline cleaving enzyme [post-proline endopeptidase: EC 3.4.21.26] was purified from lamb brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and Sephadex G-150. The purified enzyme appeared homogeneous on disc gel and sodium dodecyl sulfate (SDS) gel electrophoreses. The enzyme was most active at pH 7.0 with carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap) as a substrate and catalyzed the hydrolysis of oxytocin, vasopressin, thyrotropin releasing hormone (TRH), substance P, luteinizing hormone releasing hormone (LH-RH), and angiotensin at the carboxyl side of their proline residues, except for the Pro2-Lys3 bond in substance P. From the results of subsite mapping using synthetic peptides, five subsites, S3 to S2', for substrate interaction with the enzyme were deduced to be present, and high stereospecificity was observed at S2, S1, and S1'. The isoelectric point of the enzyme was at pH 4.9, and the molecular weights estimated by gel filtration and SDS gel electrophoresis were 74,000 and 77,000, respectively. The enzyme was markedly inhibited by diisopropylphosphoro fluoridate (DFP), carbobenzoxy-Gly-Pro-chloromethyl ketone (Z-Gly-Pro-CH2Cl), p-chloromercuribenzoate (PCMB), Hg2+, and Cu2+ ions. These enzymatic and protein chemical properties of post-proline cleaving enzyme from lamb brain closely resemble those of the lamb kidney enzyme, except for the molecular weight. In the present work, however, we decided that the molecular weight of the enzyme from lamb kidney was also 74,000, which is different from that reported previously (J. Biol. Chem. 251, 7593 (1976) but is in accord with the value of post-proline cleaving enzyme from lamb brain.

Degradation of bradykinin and its metabolites by rat brain synaptic membranes.

Bradykinin (BK) (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) was degraded by rat brain synaptic membranes at a rate comparable to that found for Met-enkephalin, but approximately 40 times the rate for vasopressin and oxytocin. The catabolic pathway for BK and its metabolites was elucidated through the use of high performance liquid chromatography for metabolite identification and peptidase inhibitors for blocking specific cleavage sites. BK was hydrolyzed at three sites: at the -Phe5-Ser6- bond by metalloendopeptidase 24.15, at the -Pro7-Phe8- bond by an apparently novel peptidyl dipeptidase, and at the -Phe8-Arg9 bond by a carboxypeptidase B-like enzyme. Each enzyme contributed about equally to BK degradation under the assay conditions used. Some of the resulting metabolites were further hydrolyzed: BK(1-8) to BK(1-7) + Phe by a DFP inhibitable prolyl carboxypeptidase-like enzyme, BK(1-8) to BK(1-5) + BK(6-8) by metalloendopeptidase 24.15, BK(1-7) slowly to BK(1-5) by a second peptidyl dipeptidase which was captopril inhibited, and Phe-Arg to Phe + Arg by a bestatin-inhibited dipeptidase. A number of properties of the individual enzymes were determined including sensitivity to a variety of peptidase inhibitors. These results provide a starting point for investigating the potential physiological role of each enzyme in BK function in the brain.

Amastatin potentiates the behavioral effects of vasopressin and oxytocin in mice.

Vasopressin and oxytocin cause behavioral excitation after intracerebroventricular injection in mice. This effect is short-lasting, suggesting that the peptides are rapidly inactivated in the brain. Co-injection of microgram amounts of amastatin, an aminopeptidase inhibitor, prolonged the effect of both vasopressin and oxytocin. Amastatin did not induce large vasopressin-like behavioral effects by itself, nor did it significantly potentiate the action of 1-deamino[1,6-dicarba, 8-arginine] vasopressin (Asu-AVP), an analog that lacks the N-terminal amino group. The effect of Asu-AVP, but not that of vasopressin, was potentiated by phosphoramidon, an inhibitor of neutral metalloendopeptidase ("enkephalinase A"). These results support previous suggestions that vasopressin and oxytocin are inactivated mainly by aminopeptidase action following intracerebroventricular injection.

Behavioral alterations induced by substance P, bombesin, and related peptides in mice.

Bombesin, substance P and several structurally related peptides cause excessive grooming behavior after intracerebroventricular injection in mice. The present study describes the behavioral characteristics of these effects after acute administration. Substance P caused an elevation of grooming behavior which was short-lasting (less than 15 minutes), while bombesin induced both grooming and scratching behavior with a duration of action of about 2.5 hours. After repeated injections of high doses of either bombesin or a metabolically stable substance P analog, no tolerance-formation to these peptide-induced effects could be observed. Morphine partially antagonized bombesin-induced behaviors at a dose of 7.5 mg/kg subcutaneously while the same dose did not attentuate substance P-induced grooming. These results suggest that the behavioral changes induced by substance P and bombesin are mediated by distinct mechanisms. The lack of tolerance formation, together with the partial antagonism by morphine, suggests that the bombesin-induced behaviors may be related to a stimulation of nociceptive mechanisms.

Des-Arg9-bradykinin metabolism in patients who presented hypersensitivity reactions during hemodialysis: role of serum ACE and aminopeptidase P.

Bradykinin (BK) has been proposed as the principal mediator of hypersensitivity reactions (HSR) in patients dialyzed using negatively charged membranes and concomitantly treated with angiotensin-converting enzyme (ACE) inhibitors. We investigated the metabolism of exogenous BK added to the sera of 13 patients dialyzed on an AN69 membrane with a history of HSR (HSR+ patients) and 10 others who did not present such a reaction (HSR- patients) while dialyzed under the same conditions. No significant difference in the t1/2 of BK was found between the patient groups. However, the t1/2 of generated des-Arg9-BK was significantly increased (2.2-fold) in HSR+ patients compared to HSR-subjects. Preincubation of the sera with an ACE inhibitor (enalaprilat) significantly increased the t1/2 of both BK and des-Arg9-BK in both groups. There was no significant difference between the groups with respect to the t1/2 of BK, but there was a significantly greater increase (3.8-fold) in the t1/2 of des-Arg9-BK in HSR+ patients compared to HSR-subjects. The level of serum aminopeptidase P (APP) activity showed a significant decrease in the HSR+ sera when compared to HSR-samples. In HSR- and HSR+ patients, a significant inverse relation (r2 = 0.6271; P < 0.00005) could be calculated between APP activity and des-Arg9-BK t1/2. In conclusion, HSR in hemodialyzed patients who are concomitantly treated with a negatively charged membrane and an ACE inhibitor can be considered as a multifactorial disease in that a decreased APP activity resulting in reduced degradation of des-Arg9-BK may lead to the accumulation of this B1 agonist that could be responsible, at least in part, for the signs and symptoms of HSR.

Vascular catabolism of bradykinin in the isolated perfused rat kidney.

Kinins in the circulation are rapidly metabolized by several different peptidases. The purpose of this study was to evaluate the contribution of membrane-bound peptidases to kinin metabolism in the renal circulation. Experiments were performed in vitro, in isolated rat kidneys perfused at a constant flow rate (8 ml/min) with Tyrode's solution. The effects of peptidase inhibitors were evaluated on the functional vasodilator response caused by bradykinin (30 nM) or [Tyr(Me)(8)]bradykinin (10 nM) via activation of bradykinin B2 receptors in kidneys precontracted with prostaglandin F2alpha. Angiotensin converting enzyme inhibitors, enalaprilat (3 microM), ramiprilat (1 microM) or lisinopril (1 microM), increased the bradykinin-induced renal vasodilation by 40% or more. Inhibitors of neutral endopeptidase (thiorphan or phosphoramidon, 10 microM), basic carboxypeptidase (DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid or MGTPA, 10 microM) and aminopeptidase P (apstatin, 20 microM) however did not enhance the renal vasodilator response elicited by kinins, whatever tested alone or in the presence of lisinopril. These findings indicate that angiotensin converting enzyme is the major peptidase whose inhibition potentiates the renal bradykinin B2 receptor mediated vasodilator response of kinins. The relative contribution in this potentiation of inhibition of kinin inactivation and of cross-talk of angiotensin converting enzyme with bradykinin B2 receptor remains however to be clarified.

Potentiation of the pro-inflammatory effects of bradykinin by inhibition of angiotensin-converting enzyme and aminopeptidase P in rat paws.

The influence of some peptidase inhibitors on oedema and plasma extravasation induced by bradykinin and carrageenan in rat paw was evaluate. Bradykinin-induced oedema in normal rats was increased by o-phenanthroline (3.10(-2) M), by captopril (10(-6) M to 10(-4) M), by lisinopril (10(-6) M to 10(-4), or by lisinopril (10(-5) M) in combination with apstatin (8.10(-5) M or 1.4 10(-4) M). It was not modified by phosphoramidon (10(-6) M to 10(-5) M) and by diprotin A (10(-3) M). It was increased by mergepta at high concentrations (2.10(-4) M). Mergepta did not increase the potentiating effect of captopril. Carrageenan-oedema in normal rats was increased by captopril (10(-5) M), lisinopril (10(-5) M) and apstatin (1.4 10(-4) M. It was not modified by mergepta (10(-4) M), phosphoramidon (10 (-5) M) and diprotin A (109-3) M). Des-Arg1-bradykinin and Des-Arg9-bradykinin have low oedema-promoting effects. Captopril (10(-5) M) increased the effects of bradykinin but not those of carrageenan in kininogen-deficit Brown Norway rats. Angiotensin-converting enzyme and aminopeptidase P appear to be main kinin-inactivating enzymes in rat paws. Carboxypeptidase N, neutral endopeptidase 24.11 and dipeptidyl(amino)peptidase IV do not play a significant role in this inactivation.

Influence of several peptidase inhibitors on the pro-inflammatory effects of substance P, capsaicin and collagenase.

Injection of substance P (SP) in a rat hindpaw induced extravasation of 125I-labelled albumin in both hindpaws and salivation. Intravenous injection of SP dose-dependently increased vascular permeability. This latter effect was increased in rat paws by captopril, an inhibitor of angiotensin-converting enzyme (ACE), administered locally in combination with diprotin A, an inhibitor of an dipeptidyl(amino)peptidase IV (DAP IV) or phosphoramidon, an inhibitor of neutral endopeptidase (NEP). The increase in permeability induced by SP was inhibited by RP 67580, a NK-1-receptor antagonist. Intravenous injection of capsaicin induced labelled albumin extravasation in rat paws. This effect was increased by combination of captopril with diprotin A or phosphoramidon, but not by captopril associated with amastatin, an inhibitor of aminopeptidase M (AmM). It was suppressed by RP 67580. Injection of collagenase in rat paws triggered a swelling and a local plasma exudation. These responses were reduced by RP 67580 but not by RP 68651, its inactive enantiomer. They were increased by combination of captopril with diprotin A or phosphoramidon in normal rats. The potentiating effects of captopril and diprotin A were suppressed by RP 67580 in normal rats but did not develop in kininogen-deficient rats. The oedema induced by collagenase was also increased by lisinopril, another ACE inhibitor, administered locally in combination with apstatin, an inhibitor of aminopeptidase P (AmP). In rats pretreated by methysergide, collagenase-induced oedema was reduced and can be increased by captopril, by lisinopril, administered alone or by lisinopril associated with apstatin. It is concluded that SP is mainly inactivated in rat paws by ACE, DAP IV and NEP. In collagenase-induced oedema, a low amount of SP would be released from afferent nerve terminals by bradykinin formed in low amounts. Bradykinin is inactivated in rat paws by ACE and AmP. In collagenase-oedema, the pro-inflammatory effects of bradykinin are concealed by the effects of the other mediators.

Factors involved in the inactivation of vasopressin after intracerebroventricular injection in mice.

Behavioral excitation induced by intracerebroventricularly administered vasopressin in mice is very short-lasting, suggesting a half-life of the injected peptide of only a few minutes. The results of the present study suggest that vasopressin and related peptides are too hydrophilic to penetrate lipid membranes readily by passive diffusion and that passive diffusion from the extracellular space into cells or the bloodstream is an unlikely mechanism of inactivation. Pharmacological desensitization (tachyphylaxis) occurs after higher doses, but does not seem to be the major factor responsible for the short duration of action. Some deaminoanalogs of vasopressin, however, show a prolonged action, suggesting that degradation by (an) aminopeptidase(s) is a major route of inactivation. These results also suggest that vasopressin-degrading aminopeptidases are accessible from the extracellular space.

Minireview. Peptides and the blood-brain barrier.

Most neuropeptides are known to occur both in the central nervous system and in blood. This, as well as the occurrence of central nervous peptide effects after peripheral administration, show the importance of studying the relationships between the peptides in the two compartments. For many peptides, such as the enkephalins, TRH, somatostatin and MIF-1, poor penetration of the blood-brain barrier was shown. In other cases, including beta-endorphin and angiotensin, peptides are rapidly degraded during or just after their entry into brain or cerebrospinal fluid. Some peptides, such as insulin, delta-sleep-inducing peptide, and the lipotropin-derived peptides, enter the cerebrospinal fluid to a slight or moderate extent in the intact form. Many peptide hormones, such as insulin, calcitonin and angiotensin, act directly on receptors in the circumventricular organs, where the blood-brain barrier is absent. Oxytocin, vasopressin, MSH, and an MSH-analog alter the properties of the blood-brain barrier, which may result in altered nutritient supply to the brain. In conclusion, the diffusion of most peptides across the brain vascular endothelium seems to be severely restricted. There are, however, several alternative routes for peripheral peptides to act on the central nervous system. The blood-brain barrier is a major obstacle for the development of pharmaceutically useful peptides, as in the case of synthetic enkephalin-analogs.

Correlations of serotonin and its metabolites in individual rat pineal glands over light:dark cycles and after acute light exposure.

Profiles of pineal indolealkylamines were estimated by high performance liquid chromatography and were correlated in individual glands of male rats sacrificed over several light:dark cycles and after acute exposure to light at night. A significant and positive correlation of 5HIAA vs 5HT in individual glands over both normal and experimental lighting conditions suggested that oxidative deamination is not a major factor in photic regulation of pineal 5HT levels and that the formation of 5HIAA is dependent on substrate availability. Regression analysis of other indole constituents revealed that there was a positive and significant correlation between 5HT vs N-acetylserotonin, but not between 5HT vs melatonin and N-acetylserotonin vs melatonin in individual glands during the dark phase of a light:dark cycle. We propose that this effect may be related to a pulsatile release of melatonin into the blood stream and is the result of sampling glands at different stages in the storage/release of melatonin.