NEURAMIDE STIMULATES ADRENOCORTICOTROPIN BUT NOT PROLACTIN RELEASE FROM RAT PITUITARY

NEURAMIDE STIMULATES ADRENOCORTICOTROPIN BUT NOT PROLACTIN RELEASE FROM RAT PITUITARY

Pharmacological Research, Vol. 34, No. 5/6, 1996 NEURAMIDE STIMULATES ADRENOCORTICOTROPIN BUT NOT PROLACTIN RELEASE FROM RAT PITUITARY LUIGI BRUNETTI...

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Pharmacological Research, Vol. 34, No. 5/6, 1996

NEURAMIDE STIMULATES ADRENOCORTICOTROPIN BUT NOT PROLACTIN RELEASE FROM RAT PITUITARY LUIGI BRUNETTI, ENZO RAGAZZONI, GIANCARLO FOLCHITTO* and MICHELE VACCA† Department of Pharmacology Catholic University, *DIFA Italy, Bergamon, Pomezia, Rome and †Institute of ‘Scienza del Farmaco’ G. D’Annunzio University, Chieti Accepted 20 September 1996 Neuramide (NMD), a substance found in crude preparations of porcine stomach extract, is a viral inhibitor that also has putative immunostimulatory effects. The effects of NMD on stresshormone (ACTH and prolactin—PRL) release were assessed in in vivo and in vitro studies. In the former, blood levels of corticosterone and PRL were measured in NMD-treated male rats. In vitro experiments were performed to evaluate the effects of NMD and three of its fractions (obtained with high performance liquid chromatography) on ACTH and PRL release from perfused rat pituitary slices. NMD increased plasma corticosterone levels in vivo and produced dose-dependent increases in in vitro pituitary release of ACTH. No effects on PRL secretion were observed in vivo or in vitro. The stimulatory effects on ACTH release were caused by the NMD fraction with a molecular weight of >5000<10000 Da. 1996 The Italian Pharmacological Society KEY WORDS: neuramide, ACTH, PRL, corticosterone.

INTRODUCTION Neuramide (NMD) is a porcine stomach-derived polypeptide mixture with an apparently wide spectrum of antiviral activity. In vivo studies have shown that NMD is effective in the treatment of vesicular stomatitis and infections caused by varicella and Herpes zoster viruses [1]. It has also been shown to inhibit in vitro replication in adsorbed influenza A viruses [2] and a number of other viruses as well [3, 4]. As an aqueous solution with a mean concentration of 61 mg ml−1 of a peptide weighing <10000 Da, NMD has been found to exert two distinct effects against herpes and other viruses, each of which is related to a natural viral inhibitor. The first, known as CVI (cellular viral inhibitor), has a molecular weight of less than 2500 Da and acts outside the cell to prevent viral absorption through the cell membrane [2–4]. The second factor acts after viral penetration to prevent replication [5, 6]. The antiviral activity of NMD is measured in viral replication-inhibiting units. Various attempts have been made to characterize other properties of NMD, including the non-specific forms of defense against viral infections that appear to be exerted by its CVI. The drug has been shown to possess immunostimulatory properties reflected by its enhancement of phytohemagglutinin (PHA)-induced

Correspondence to: Prof. Michele Vacca, Institute of ‘Scienza del Farmaco’ G. D’Annunzio University, Via dei Vestini, 31, 66013 Chieti, Italy. 1043–6618/96/120269–05/$25.00/0

proliferation in mixed lymphocyte cultures and by the increases it produces in immunoglobulin production in pokeweed mitogen-stimulated lymphocyte cultures [7]. In contrast, NMD does not appear to stimulate or inhibit NK cells, whose effects on target cells do not require previous cellular proliferation. In light of the immune-mediated effect of the hepatitis B virus, the seemingly positive effects of NMD in acute infections of this type also suggest that the drug might have stimulatory effects on the immune system, which is involved not only in the liver damage provoked by the virus, but also in its resolution [8]. It is also important to recall that immunodepressive phenomena are characteristic of various viral infections, including varicella, which appears to be sensitive to NMD [1]. Martini et al. [9] found that NMD exerts a peripheral analgesic effect in animals that does not involve opioid receptors, and a number of groups have demonstrated its antiinflammatory effects in neuritis and radiculitis [10–13]. Size-exclusion high-performance liquid chromatography (HPLC) analysis has shown that NMD is composed of several fractions with different molecular weights (MW), each of which appears to exert a distinct activity on its own [14]. Consecutive MW sieving using Amicon (types UM, YM and PM) and Waters membranes with various pore sizes has been used to separate fractions with MWs of <1000, 500–1000 and <500 Da [14]. Anti-influenza virus activity was found to be peculiar to the <1000 Da fraction [2], while immunostimulatory activity (i.e. 1996 The Italian Pharmacological Society

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potentiation of PHA-induced human lymphocyte proliferation) was displayed by four fractions with MW− <5000 Da. It is well known that there is significant interaction between the immune and endocrine systems. A number of immune cell-derived molecules are known to exert endocrine effects, and the importance of neuroendocrine modulation of immune/inflammatory responses has become increasingly evident over the past decade [15–17]. In view of the immunostimulatory and anti-inflammatory effects of NMD, the present study was conducted to investigate the possible effects of this drug and its various fractions on the major components of the hypothalamic-pituitary-adrenal (HPA) axis, which is known to play an active role in both inflammatory processes and immunomodulation. To this end, in vivo experiments were conducted to assess the effects of NMD treatment on prolactin (PRL) and corticosterone secretion in rats. In vitro studies were also conducted to measure the release of adrenocorticotropin (ACTH) and PRL from rat pituitary slices after exposure to NMD and three of its fractions. Prolactin assays were included in this study in part because of its own immunomodulatory effects [18], but also because it is co-released with ACTH during stress reactions [17] and can thus be used as an additional index of the possible stimulatory effects of NMD on the HPA axis.

MATERIALS AND METHODS

Animals Adult male Wistar rats (200–250 g) were kept for one week or more in a thermoregulated environment (23±1°C) with automatic light control that provided 14 h of light (06:00–20:00) followed by 10 h of darkness/day. Food and water were available ad libitum. The animals were killed by decapitation. For in vivo experiments trunk blood was collected and centrifuged and the plasma promptly frozen. For in vitro experiments, the anterior pituitaries were rapidly removed and perfused as described below.

Superfusion of pituitary slices Each gland was sliced into 0.1–0.2 mm-thick sections and placed inside cylindrical plexi-glass chambers (diameter 5 mm; height 20 mm) mounted inside a 37°C water jacket. The perfusion procedure has been described in detail elsewhere [19]. Briefly, the tissue slices were positioned on a nylon mesh (pore size 25 µM) at the bottom of the chambers, and perfused with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 0.012 M NaHCO 3, 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 0.01 M TES (2-{[tris-(hydroxymethyl) methyl] amino} ethanesulfonic acid), and adjusted to pH 7.5 with 5 M NaOH. A peristaltic pump was used to circulate the perfusion medium through the system

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at a flow rate of 0.4 ml min−1. To avoid mechanical disturbance of the hypothalamic slices, the medium was introduced from above and warmed to a temperature of 37°C as it ran down the sides of the chamber. Eight superfusion lines could be run simultaneously, with one untreated line used to monitor basal secretion. The effluents, collected in 5-min fractions in tubes containing 100 µl 1% bovine serum albumin in phosphate-buffered saline, were stored at −40°C until assayed. Baseline values were determined after a 120-min preincubation. Test solutions were introduced by means of a 4-way valve to avoid interruption of the flow. During superfusion, the tissue slices were completely immersed in approximately 400 µl of medium.

Drugs In vivo experiments. NMD was supplied by DifaCooper Pharmaceuticals (Caronno Pertusella, Va, Italy) in 1.3-ml vials containing 56–68 mg ml−1 peptides weighing <10000 Da (mean concentration: 61 mg ml−1), 190–210 µg free aminoacids and 9–12 mg carbohydrates. The drug was administered by intramuscular injection at increasing doses (0.1–1.0–5.0 ml kg−1) 24, 8, 6, 4 and 2 h before the animals were sacrificed. Eight to ten rats were used to test each dose and injection time. Because handling and injection can increase plasma corticosterone levels, controls were performed for each dose/injection–time using matched-volume saline injections. In vitro experiments. NMD was added to the perfusion medium to produce dilutions of 1:10, 1:50 and 1:100. In order to identify specific NMD fraction(s) with possible endocrine effects, additional experiments were performed with three NMD fractions: <1000 Da, >1000 Da and <5000 Da and <10000 Da obtained by HPLC as previously described [14]. Each fraction was tested at concentrations of 1:50, 1:100, 1:500 and 1:1000.

Hormone assays ACTH and PRL levels (in trunk blood collected for in vivo experiments and in perfusion effluents for in vitro studies) were measured by radioimmunoassay, using kits supplied by the NIDDK Rat Pituitary Hormone Distribution Program. The technique used for the ACTH assays was based on the data of Rees et al. [20] and Matsuyama et al. [21]. In in vitro experiments, corticosterone levels were measured in trunk blood with a radioimmunological method developed in our laboratory [22].

Statistical methods The results of the in vivo experiments were expressed as arithmetic means±standard errors (SE) of the means for each group. The data were then subjected to analysis of variance and the Student-

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PRL (ng ml )

–1

–1

ACTH (pg ml )

Neuramide 250 200 150 100 50 0

100 Neuramide

80 60 40 20

20

40

60

80

100

0

20

Time (min)

40

60

80

100

Time (min)

Fig. 2. Effect of neuramide on rat pituitary secretion of PRL in vitro. Arrows indicate the interval during which tissue slices were exposed to neuramide. The figure is representative of one of three or more similar experiments. s, Control; j, Neuramide 1:10; m, Neuramide 1:50; e, Neuramide 1:100.

Newman-Keul test for multiple comparisons among groups. Differences between group means were considered significant when P<0.05.

Neuramide

RESULTS As shown in Table I, the highest dose of NMD tested in the in vivo experiments (5 ml kg −1, corresponding to approximately 300 mg kg−1 peptides) produced significant increases over baseline in plasma corticosterone levels in trunk blood when given 6 h or less before being killed. This effect peaked approximately 4 h after administration. None of the test doses of NMD had any effect on plasma PRL levels. In the in vitro experiments, NMD produced dosedependent increases in the release of ACTH by perfused anterior pituitary slices (Fig. 1) that peaked approximately 5 min after addition of the drug and persisted for the entire 20-min period of exposure. None of the tested dilutions had any effect on PRL secretion (Fig. 2). Dose-dependent increases in in vitro ACTH release were also observed shortly after the addition to the

–1

ACTH (pg ml )

Fig. 1. Effect of neuramide on rat pituitary secretion of ACTH in vitro. Arrows indicate the interval during which tissue slices were exposed to neuramide. The figure is representative of one of three or more similar experiments. s, Control; j, Neuramide 1:10; m, Neuramide 1:50; e, Neuramide 1:100.

500 400 300 200 100

20

0

40

60

80

Fig. 3. Effect of the >5000<10000 Da fraction of neuramide on rat pituitary secretion of ACTH in vitro. Arrows indicate the interval during which tissue slices were exposed to the neuramide fraction (at dilutions of 1:50, 1:100, 1:500 and 1:1000). The figure is representative of one of three or more similar experiments. s, 1:50; j, 1:100; n, 1:500; ◆, 1:1000.

perfusion medium of the NMD fraction with MW> 5000<10000 Da. (Fig. 3). None of the other fractions had any effect on this release at any of the dilutions tested (Fig. 4). None of the fractions, including the

Table I Time course of plasma corticosterone and PRL concentrations following graded doses of intramuscular (i.m.) neuramide ‘in toto’ injections Treatment (ml kg−1 )

2h

4h

6h

8h

24 h

36±2.4 31±2.5 45±4.1 107±9.8**

47±3.2 36±3.4 56±5.7 153±12**

41±3.2 36±2.1 37±2.8 72±7.4*

46±5.1 — — 44±4.4

29.4±2.9 — — 31±2.5

8±0.4 7.3±0.6 7.6±0.8 6.9±0.9

8.4±0.7 8.3±0.7 8.4±0.5 9.2±0.8

7±0.3 6.9±0.7 6.4±0.6 8.8±0.9

8.2±1.9 — — 11.4±1.9

7.5±1.6 — — 7.3±1.6

−1

Plasma corticosterone (ng ml ) Saline NMD 0.1 NMD 1 NMD 5 PRL (ng ml−1 ) Saline NMD 0.1 NMD 1 NMD 5

100

Time (min)

*P<0.05 vs controls. **P<0.01 vs controls. n=8–12 animals/each experimental group.

ACTH (pg ml–1)

272

Pharmacological Research, Vol. 34, No. 5/6, 1996 200 190 180 170 160 150 140 130 120 110 100 90

Neuramide

0

20

40 60 Time (min)

80

100

Fig. 4. Effect of the <1000 Da and >1000<5000 Da fractions of neuramide on rat pituitary secretion of ACTH in vitro. Arrows indicate the interval during which tissue slices were exposed to the neuramide fraction. Only the highest dilution tested (1:50) is shown. The figure is representative of one of three or more similar experiments. s, Fraction <1000 Da; d, Fraction >1000<5000 Da.

80

–1

PRL (ng ml )

100

Neuramide

60 40 20 0

20

40 60 Time (min)

80

effect that has been observed with other immunomodulating substances [23, 24]. In previous studies of ours, neither NMD nor any of its fraction had any direct effects on rat adrenal gland slices (unpublished data). Nonetheless, the results of our in vitro studies clearly show that the drug does exert a direct stimulatory effect on pituitary ACTH release. In the absence of structural data on the >5000 Da fraction, it is difficult to speculate on the possibility of a receptor-mediated cross-effect at the hypophyseal level between this fraction and known ACTH secretagogues. While the in vitro immunostimulatory and antiviral effects of NMD have been attributed to fractions with MWS of <5000 Da [6], our findings indicate that the drug’s ability to activate the HPA axis depends upon a fraction with MWs of >5000 and <10000 Da. At present it is difficult to relate this latter effect to any of the reported clinical effects of NMD, with the possible exceptions of the analgesic activity documented in experimental animals and shown to be unrelated to endogenous opioids or opioid receptors [9] or its antiinflammatory effects [25], which have yet to be linked to any specific fraction of the drug.

100

ACKNOWLEDGEMENTS

Fig. 5. Effect of all tested fractions of neuramide on rat pituitary secretion of PRL in vitro. Arrows indicate the interval during which tissue slices were exposed to the neuramide fraction. Only the highest dilution tested (1:50) is shown. The figure is representative of one of three or more similar experiments. s, Fraction <1000 Da; j, Fraction >1000<5000 Da; m, Fraction >5000 <1000 Da.

The authors are grateful for the valuable comments and suggestions made by Prof. Paolo Preziosi during the preparation of this paper. We are also grateful to Ms Cinzia Molino for careful typing of the text and to NIDDK for supplying PRL and ACTH RIA kits.

one with MW >5000<10000 Da, affected PRL release (Fig. 5).

REFERENCES

DISCUSSION In addition to its direct immunostimulatory and antiviral effects [2–5], NMD clearly appears, on the basis of our findings, to activate the HPA axis via direct stimulation of pituitary ACTH release. Our in vitro data indicate that this effect is produced by the fraction with a MW of >5000 Da and <10000 Da. Neither NMD itself nor any of the fractions tested had any effect on PRL secretion. In vitro ACTH release was significantly enhanced for the entire 20-min period in which NMD or its >5000<10000 Da fraction was present in the perfusion liquid, while increased plasma levels of corticosterone were found up to 6 h after administration of the drug in the in vivo experiments. These findings suggest that NMD or its >5000 Da fraction might also activate a peripheral source of ACTH, an

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