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Neuropeptide K and Neurokinin A Stimulate CRH and ACTH Release by Rat Adrenal Medulla in Vitro
Peptides, Vol. 18, No. 4, pp. 487–490, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(97)00066-1
Neuropeptide K and Neurokinin A Stimulate CRH and ACTH Release by Rat Adrenal Medulla In Vitro GIUSEPPINA MAZZOCCHI, LUDWIK K. MALENDOWICZ, GIUSEPPE GOTTARDO AND GASTONE G. NUSSDORFER 1 Department of Anatomy, University of Padua, I-35121 Padua, Italy Received 21 October 1996; Accepted 17 December 1996 MAZZOCCHI, G., L. K. MALENDOWICZ, G. GOTTARDO AND G. G. NUSSDORFER. Neuropeptide K and neurokinin A stimulate CRH and ACTH release by rat adrenal medulla in vitro. PEPTIDES 18(4) 487–490, 1997.—Tachykinins are a family of peptides that are able to modulate the activity of the hypothalamo–pituitary CRH–ACTH system. Mammalian tachykinins include neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), and substance P (SP). We investigated by RIA the effects of tachykinins on the release of CRH and ACTH by rat adrenal medulla in vitro. NKA and NPK concentration-dependently enhanced the release of both CRH and ACTH, NPK being more active than NKA. NKB exerted only a minor stimulatory action exclusively on CRH release, and SP was ineffective. The stimulatory effect of both NKA and NPK on ACTH release was blocked by the CRH receptor antagonist a-helical-CRH, thereby suggesting that the increase in ACTH secretion is consequent to the stimulation of CRH release. These findings indicate that NKA and NPK are stimulators not only of the central (hypothalamo– pituitary), but also of the peripheral (intramedullary) branch of the CRH–ACTH system. q 1997 Elsevier Science Inc. Neurokinin A
Neuropeptide K
Adrenal medulla
CRH
Rat
METHOD
MAMMALIAN tachykinins are a family of neuropeptides including neurokinin B (NKB), neurokinin A (NKA), substance P (SP), structurally related to NKA and encoded by the same gene, and neuropeptide K (NPK), which is the n-terminally extended form of NKA. Tachykinins act via three subtypes of receptors, named NK1 , NK2 , and NK3 , which show the highest affinity for SP, NKA and NPK, and NKB, respectively (12). Tachykinins are widely distributed in the central nervous system, and are able to regulate the activity of the hypothalamo–pituitary–adrenal axis (10,13). Many lines of evidence indicate that rat adrenal medulla contains and releases CRH and ACTH immunoreactivities (2,3,9,19), and findings are available that in this species an intramedullary CRH–ACTH system is operative, duplicating that existing at the hypothalamo–pituitary level and involved in the paracrine stimulation of glucocorticoid secretion [ for review, see (20)]. In a previous study, we provided an indirect demonstration that NPK exerts a marked glucocorticoid secretagogue effect in rats, acting not only on the central, but also on the peripheral (intramedullary), branch of the CRH–ACTH system (17). It therefore seemed worthwhile to investigate whether the various mammalian tachykinins are able to affect CRH and ACTH release by rat adrenal medulla in vitro.
1
ACTH
Adult male Wistar rats, weighing 200 { 20 g, were purchased from Charles River (Como, Italy). They were decapitated, and their adrenal glands promptly excised and freed of pericapsular fat. The protocol of the experiment was approved by the Animal Research Commettee of the Padua University. NKA, NKB, SP, and NPK were purchased from Peninsula Laboratories (Merseyside, UK), a-helical-CRH(9–41) ( aCRH), an antagonist of CRH receptors (21), from Sigma Chemical Co. (St. Louis, MO), and Medium 199 from DIFCO (Detroit, MI). RIA kits for CRH and ACTH were obtained from Peninsula. Adrenal glands were decapsulated and hemisected; then adrenal halves were enucleated, under the dissecting microscope, for removal of the medulla. Medullary fragments (about 10 mg/ adrenal pair) were put in 2 ml of Medium 199 and Krebs–Ringer bicarbonate buffer with 0.2% glucose. They were incubated in replicates of 30 each, as follows: (i) increasing concentration of tachykinins (from 10 012 to 10 06 M); and (ii) 10 06 M NKA or NPK in the presence or absence of 10 06 M a-CRH. The incubation was carried out for 120 min in a shaking bath at 377C in an atmosphere of 95% O2 /5% CO2 . Incubation media were defatted with 3 vol of petroleum ether at 47C, and aqueous extracts
Requests for reprints should be addressed to Prof. Gastone G. Nussdorfer, Department of Anatomy, Via Gabelli 65, I-35121 Padova, ltaly.
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were evaporated to dryness. The recovery was about 60 10% SD and 80 12% SD for CRH and ACTH, respectively (19). The residues were redissolved (pooled 5 by 5 to obtain six samples for each experimental point) in 1 ml PBS, and stored at 0307C until RIA assay. CRH and ACTH were extracted on SEP-Pak C18 cartridge (Waters, Milford, MA) and then eluted and purified by reversephase HPLC (19,23,25). CRH and ACTH concentrations were determined by RIA. CRH (human, rat) RIA kit: sensitivity, 2.5 fmol/ml. Cross-reactivity: CRH (human, rat), 100%; CRH (ovine), 0.1%; sauvagine and GH-RH (rat), less than 0.001%. No cross-reactivity was shown for a-CRH. Intra- and interassay variations were 6.2% and 8.3%, respectively. ACTH (rat) RIA kit: sensitivity, 1 fmol/ml. Cross-reactivity: ACTH (human, rat), 100%; ACTH(1–24), 0.01%; other ACTH fragments and pituitary hormones, 0%. Intra- and interassay variations were 5.7% and 7.4%, respectively. Data were expressed as means { SE, and their statistical comparison was performed by ANOVA, followed by the Multiple Range Test of Duncan. A value of p õ 0.05 was considered significant. RESULTS
NKA and NPK concentration-dependently stimulated rat adrenal medullary tissue to release CRH [NKA: F (5, 25) Å 3.62, p õ 0.01; NPK: F (5, 25) Å 4.89, p õ 0.01] (Fig. 1) and ACTH [NKA: F (5, 25) Å 2.64, p õ 0.05; NPK: F(5, 25) Å 4.02, p õ 0.01] (Fig. 2). In both cases, NPK appeared to be more effective FIG. 2. Effect of tachykinins of ACTH release by rat adrenal medulla tissue. Values are means { SE (n Å 6). /p õ 0.05 and * p õ 0.01 vs. baseline (B).
than NKA. NKB elicited a moderate rise in the CRH, but not ACTH secretion, whereas SP was ineffective (Figs. 1 and 2). a-CRH (10 06 M) abolished the 10 06 M NPK- and NKAevoked rises in ACTH release, without altering basal secretion per se (Fig. 3). a-CRH (10 06 M) did not affect CRH responses to tachykinins (Fig. 4). DISCUSSION
FIG. 1. Effect of tachykinins on CRH release by rat adrenal medulla tissue. Values are means { SE (n Å 6). / p õ 0.05 and * p õ 0.01 vs. baseline (B).
NKA and NPK have been previously found to evoke a moderate and intense activation, respectively, of the hypothalamo– pituitary adrenal axis in rats (10,15). Subsequently, NPK was shown to enhance glucocorticoid secretion of rat adrenal slices containing chromaffin tissue, but not of dispersed adrenocortical cells. The secretagogue effect of NPK was blocked by both aCRH and the ACTH receptor antagonist corticotropin-inhibiting peptide, thereby suggesting that it may be indirectly mediated by the activation of the intramedullary CRH–ACTH system (17). Our present findings are in keeping with this hypothesis, because they clearly show that both NKA and NPK significantly enhance the release of both CRH and ACTH by rat adrenal medulla. The present finding that NKA is less effective than NPK appears to be in keeping with the hypothesis that NPK is the endogenous biologically active tachykinin, NKA being a sequential degradation product (10): an hypothesis that agrees with the lack of effect of NKA on hypophyseal ACTH release (8,22). The possibility that NKA and NPK stimulate the corelease of CRH and ACTH can be easily ruled out, inasmuch as a-CRH blocks the effect of both tachykinins on ACTH secretion, without
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FIG. 3. Effect of -CRH (10–6 M) on basal and NKA (10 06 M)- or NPK (10 06 M )-stimulated ACTH release by rat adrenal medulla tissue. Values are means SE (n Å 6). *p õ 0.01 vs. the respective basal value (B); A p õ 0.01 vs. the respective control group.
FIG. 4. Lack of effect of a-CRH (10 06 M ) on basal and NKA (10 06 M)- or NPK (10 06 M)-stimulated CRH release by rat adrenal medulla tissue. Values are means SE (n Å 6): * p õ 0.01 vs. the respective basal value (B).
affecting that of CRH. The possibility of a toxic nonspecific effect of a-CRH can be excluded, because this antagonist does not alter the basal release of the two peptides. Hence, it seems reasonable to conceive that the primary action of NKA and NPK is the stimulation of the release by chromaffin cells of CRH, which in turn evokes the local ACTH secretion in a paracrine or autocrine manner. This hypothesis accords well with the demonstration that adrenal chromaffin cells are provided with CRH receptors (1,7,24). The presence of tachykinin immunoreactivities in the adrenal medulla of several mammalian species, including the rat, has been demonstrated by both RIA (4–6,22,26) and immunocytochemistry (11,27). Therefore, in light of our present findings, NKA and NPK may be included in that group of intra-adrenal regulatory peptides, which activate intramedullary CRH–ACTH system, thereby indirectly stimulating glucocorticoid secretion of the cortex. At present, this group appears to include argininevasopressin, pituitary adenylate cyclase-activating polypeptide, neuromedin U8, and interleukin-1 [reviewed in (20)].
NKB and SP seem to be almost completely ineffective on the intramedullary CRH–ACTH system, which suggests that the NK2 is the receptor subtype mediating this effect of tachykinins (see Introduction). The very weak stimulatory action of NKB on CRH, but not ACTH, release by adrenal medulla remains to be investigated. It may be tentatively suggested that the effect of this tachykinin is too slight to determine a local concentration of CRH sufficient to induce a sizable ACTH release. In this connection, it is to be recalled that NKB, when systemically administered, appears to induce very doubtful effects on the pituitary ACTH release (14). The lacking of any appreciable effect of SP on the CRH and ACTH release by rat adrenal chromaffin cells agrees with the demonstration that this peptide does not affect either pituitary ACTH or glucocorticoid secretion in rats (16,18). SP has been reported to inhibit hypothalamic CRH secretion (8), a finding that could suggest this may also occur in the adrenal medulla. However, it is conceivable that, due to the very low basal rate of CRH release by chromaffin cells, the inhibitory action of SP cannot be able to elicits detectable effects.
REFERENCES 1. Aguilera, G.; Millan, M. A.; Hauger, R. L.; Catt, K. J. Corticotropinreleasing factor receptors: Distribution and regulation in brain, pituitary and peripheral tissues. Ann. NY Acad. Sci. 512:48–66; 1987. 2. Andreis, P. G.; Neri, G.; Mazzocchi, G.; Musajo, F. G.; Nussdorfer, G. G. Direct secretagogue effect of corticotropin-releasing factor on the rat adrenal cortex: The involvement of the zona medullaris. Endocrinology 131:69–72; 1992.
3. Bagdy, G.; Calogero, A. E.; Szemeredi, K.; Chrousos, G. P.; Gold, P. W. Effects of cortisol treatment on brain and adrenal corticotropin-releasing hormone (CRH) content and other parameters regulated by CRH. Regul. Pept. 31:83–92; 1990. 4. Basile, S.; Cheung, N. S.; Livett, B. G. Chromatographic evidence for the presence of multiple tachykinins in the bovine adrenal medulla J. Neurochem. 58:1584–1586; 1992.
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5. Bucsics, A.; Saria, A.; Lembeck, F. Substance P in the adrenal gland: Origin and species distribution. Neuropeptides 1:329–341; 1981. 6. Cheung, N. S.; Basile, S.; Livett, B. G. Identification of multiple tachykinins in bovine adrenal medulla using an improved chromatographic procedure. Neuropeptides 24:91–97; 1993. 7. Dave, J. R.; Eiden, L. E.; Eskay, R. L. Corticotropin-releasing factor binding to peripheral tissues and activation of the adenylate cyclaseadenosine 3 *,5 *-monophosphate system. Endocrinology 116:2152– 2159; 1985. 8. Faria, M.; Navarra, P.; Tsagarakis, S.; Besser, G. M.; Grossman, A. B. Inhibition of CRH-41 release by substance P, but not substance K, from the rat hypothalamus in vitro. Brain Res. 538:76–78; 1991. 9. Hashimoto, K.; Murakami, K.; Hattori, T.; Niimi, M.; Fijimo, K.; Ota, Z. Corticotropin-releasing factor (CRF)-like immunoreactivity in the adrenal medulla. Peptides 5:707–712; 1984. 10. Kalra, P. S.; Kalra, S. P. Neuropeptide K stimulates corticosterone release in the rat. Brain Res. 610:330–333; 1993. 11. Kuramoto, H.; Kondo, H.; Fujita, T. Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats. Histochemistry 82:507–512; 1985. 12. Maggio, J. E. Tachykinins. Annu. Rev. Neurosci. 11:13–28; 1988. 13. Malendowicz, L. K.; Markowska, A. Neuromedins and their involvement in the regulation of growth, structure and function of the adrenal cortex. Histol. Histopathol. 9:591–601; 1994. 14. Malendowicz, L. K.; Nussdorfer, G. G.; Warchol, J. B.; Markowska, A.; Macchi, C.; Nowak, K. W.; Butowska, W. Effects of neuromedin-K on the rat hypothalamo–pituitary–adrenal axis. Neuropeptides 29:337–341; 1995. 15. Malendowicz, L. K.; Warchol, J. B.; Nussdorfer, G. G.; Nowak, M.; Filipiak, K. Effect of neurokinin-A on the rat hypothalamo–pituitary–adrenal axis. Endocr. Res. 21:757–767; 1995. 16. Mazzocchi, G.; Macchi, C.; Malendowicz, L. K.; Nussdorfer, G. G. Evidence that endogenous substance-P (SP) is involved in the maintenance of the growth and steroidogenic capacity of rat adrenal zona glomerulosa. Neuropeptides 29:53–58; 1995. 17. Mazzocchi, G.; Malendowicz, L. K.; Andreis, P. G.; Meneghelli, V.; Markowska, A.; Belloni, A. S.; Nussdorfer, G. G. Neuropeptide K
18. 19.
20. 21. 22. 23.
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25. 26.
27.
enhances glucocorticoid release by acting directly on the rat adrenal gland: The possible involvement of zona medullaris. Brain Res. 661:91–96; 1994. Mazzocchi, G.; Malendowicz, L. K.; Belloni, A. S.; Nussdorfer, G. G. Adrenal medulla is involved in the aldosterone secretagogue effect of substance P. Peptides 16:351–355; 1995. Mazzocchi, G.; Musajo, F. G.; Malendowicz, L. K.; Andreis, P. G.; Nussdorfer, G. G. Interleukin-1 stimulates corticotropin-releasing hormone (CRH) and adrenocorticotropin (ACTH) release by rat adrenal gland in vitro. Mol. Cell. Neurosci. 4:267–270; 1993. Nussdorfer, G. G. Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol. Rev. 48:395–430; 1996. Rivier, J.; Rivier, C.; Vale, W. Synthetic competitive antagonists of corticotropin-releasing factor: Effect on ACTH secretion in the rat. Science 224:889–890; 1984. Saria, A.; Wilson, S. P.; Molnar, A.; Viveros, O. H.; Lembeck, F. Substance P and opiate-like peptides in human adrenal medulla. Neurosci. Lett. 20:195–200; 1980. Szemeredi, K.; Bagdy, G.; Stull, R.; Calogero, A. E.; Kopin, I. J.; Goldstein, D. S. Sympatho – adrenomedullary inhibition by chronic glucocorticoid treatment in conscious rats. Endocrinology 123:2585 – 2590; 1988. Udelsman, R.; Harwood, J. P.; Millan, M. A.; Chrousos, G. P.; Goldstein, D. S.; Zimlichman, R.; Catt, K. J.; Aguilera, G. Functional corticotropin-releasing factor receptors in the primate peripheral sympathetic nervous system. Nature 319:147 – 150; 1986. Vale, W.; Vaughan, J.; Yamamoto, G.; Bruhn, T.; Douglas, C.; Dalton, D.; Rivier, C.; Rivier, J. Assay of corticotropin-releasing factor. Methods Enzymol. 103:565 – 577; 1983. Vaupel, R.; Jarry, H.; Schlo¨mer, H. T.; Wuttke, W. Differential response of substance P-containing subtypes of adrenomedullary cells to different stressors. Endocrinology 123:2140 – 2145; 1988. Wang, J. M.; De Ridder, E. F. M. M.; De Potter, W. P.; Weyns, A. L. M. Localization of neurokinin A and chromogranin A immunoreactivity in the developing porcine adrenal medulla. Histochem. J. 26:431 – 436; 1994.
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Neuropeptide K and Neurokinin A Stimulate CRH and ACTH Release by Rat Adrenal Medulla In Vitro GIUSEPPINA MAZZOCCHI, LUDWIK K. MALENDOWICZ, GIUSEPPE GOTTARDO AND GASTONE G. NUSSDORFER 1 Department of Anatomy, University of Padua, I-35121 Padua, Italy Received 21 October 1996; Accepted 17 December 1996 MAZZOCCHI, G., L. K. MALENDOWICZ, G. GOTTARDO AND G. G. NUSSDORFER. Neuropeptide K and neurokinin A stimulate CRH and ACTH release by rat adrenal medulla in vitro. PEPTIDES 18(4) 487–490, 1997.—Tachykinins are a family of peptides that are able to modulate the activity of the hypothalamo–pituitary CRH–ACTH system. Mammalian tachykinins include neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), and substance P (SP). We investigated by RIA the effects of tachykinins on the release of CRH and ACTH by rat adrenal medulla in vitro. NKA and NPK concentration-dependently enhanced the release of both CRH and ACTH, NPK being more active than NKA. NKB exerted only a minor stimulatory action exclusively on CRH release, and SP was ineffective. The stimulatory effect of both NKA and NPK on ACTH release was blocked by the CRH receptor antagonist a-helical-CRH, thereby suggesting that the increase in ACTH secretion is consequent to the stimulation of CRH release. These findings indicate that NKA and NPK are stimulators not only of the central (hypothalamo– pituitary), but also of the peripheral (intramedullary) branch of the CRH–ACTH system. q 1997 Elsevier Science Inc. Neurokinin A
Neuropeptide K
Adrenal medulla
CRH
Rat
METHOD
MAMMALIAN tachykinins are a family of neuropeptides including neurokinin B (NKB), neurokinin A (NKA), substance P (SP), structurally related to NKA and encoded by the same gene, and neuropeptide K (NPK), which is the n-terminally extended form of NKA. Tachykinins act via three subtypes of receptors, named NK1 , NK2 , and NK3 , which show the highest affinity for SP, NKA and NPK, and NKB, respectively (12). Tachykinins are widely distributed in the central nervous system, and are able to regulate the activity of the hypothalamo–pituitary–adrenal axis (10,13). Many lines of evidence indicate that rat adrenal medulla contains and releases CRH and ACTH immunoreactivities (2,3,9,19), and findings are available that in this species an intramedullary CRH–ACTH system is operative, duplicating that existing at the hypothalamo–pituitary level and involved in the paracrine stimulation of glucocorticoid secretion [ for review, see (20)]. In a previous study, we provided an indirect demonstration that NPK exerts a marked glucocorticoid secretagogue effect in rats, acting not only on the central, but also on the peripheral (intramedullary), branch of the CRH–ACTH system (17). It therefore seemed worthwhile to investigate whether the various mammalian tachykinins are able to affect CRH and ACTH release by rat adrenal medulla in vitro.
1
ACTH
Adult male Wistar rats, weighing 200 { 20 g, were purchased from Charles River (Como, Italy). They were decapitated, and their adrenal glands promptly excised and freed of pericapsular fat. The protocol of the experiment was approved by the Animal Research Commettee of the Padua University. NKA, NKB, SP, and NPK were purchased from Peninsula Laboratories (Merseyside, UK), a-helical-CRH(9–41) ( aCRH), an antagonist of CRH receptors (21), from Sigma Chemical Co. (St. Louis, MO), and Medium 199 from DIFCO (Detroit, MI). RIA kits for CRH and ACTH were obtained from Peninsula. Adrenal glands were decapsulated and hemisected; then adrenal halves were enucleated, under the dissecting microscope, for removal of the medulla. Medullary fragments (about 10 mg/ adrenal pair) were put in 2 ml of Medium 199 and Krebs–Ringer bicarbonate buffer with 0.2% glucose. They were incubated in replicates of 30 each, as follows: (i) increasing concentration of tachykinins (from 10 012 to 10 06 M); and (ii) 10 06 M NKA or NPK in the presence or absence of 10 06 M a-CRH. The incubation was carried out for 120 min in a shaking bath at 377C in an atmosphere of 95% O2 /5% CO2 . Incubation media were defatted with 3 vol of petroleum ether at 47C, and aqueous extracts
Requests for reprints should be addressed to Prof. Gastone G. Nussdorfer, Department of Anatomy, Via Gabelli 65, I-35121 Padova, ltaly.
487 / 2x14 2345 Mp 487 Tuesday May 27 08:27 PM EL–PEP (v.18#4) 2345
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were evaporated to dryness. The recovery was about 60 10% SD and 80 12% SD for CRH and ACTH, respectively (19). The residues were redissolved (pooled 5 by 5 to obtain six samples for each experimental point) in 1 ml PBS, and stored at 0307C until RIA assay. CRH and ACTH were extracted on SEP-Pak C18 cartridge (Waters, Milford, MA) and then eluted and purified by reversephase HPLC (19,23,25). CRH and ACTH concentrations were determined by RIA. CRH (human, rat) RIA kit: sensitivity, 2.5 fmol/ml. Cross-reactivity: CRH (human, rat), 100%; CRH (ovine), 0.1%; sauvagine and GH-RH (rat), less than 0.001%. No cross-reactivity was shown for a-CRH. Intra- and interassay variations were 6.2% and 8.3%, respectively. ACTH (rat) RIA kit: sensitivity, 1 fmol/ml. Cross-reactivity: ACTH (human, rat), 100%; ACTH(1–24), 0.01%; other ACTH fragments and pituitary hormones, 0%. Intra- and interassay variations were 5.7% and 7.4%, respectively. Data were expressed as means { SE, and their statistical comparison was performed by ANOVA, followed by the Multiple Range Test of Duncan. A value of p õ 0.05 was considered significant. RESULTS
NKA and NPK concentration-dependently stimulated rat adrenal medullary tissue to release CRH [NKA: F (5, 25) Å 3.62, p õ 0.01; NPK: F (5, 25) Å 4.89, p õ 0.01] (Fig. 1) and ACTH [NKA: F (5, 25) Å 2.64, p õ 0.05; NPK: F(5, 25) Å 4.02, p õ 0.01] (Fig. 2). In both cases, NPK appeared to be more effective FIG. 2. Effect of tachykinins of ACTH release by rat adrenal medulla tissue. Values are means { SE (n Å 6). /p õ 0.05 and * p õ 0.01 vs. baseline (B).
than NKA. NKB elicited a moderate rise in the CRH, but not ACTH secretion, whereas SP was ineffective (Figs. 1 and 2). a-CRH (10 06 M) abolished the 10 06 M NPK- and NKAevoked rises in ACTH release, without altering basal secretion per se (Fig. 3). a-CRH (10 06 M) did not affect CRH responses to tachykinins (Fig. 4). DISCUSSION
FIG. 1. Effect of tachykinins on CRH release by rat adrenal medulla tissue. Values are means { SE (n Å 6). / p õ 0.05 and * p õ 0.01 vs. baseline (B).
NKA and NPK have been previously found to evoke a moderate and intense activation, respectively, of the hypothalamo– pituitary adrenal axis in rats (10,15). Subsequently, NPK was shown to enhance glucocorticoid secretion of rat adrenal slices containing chromaffin tissue, but not of dispersed adrenocortical cells. The secretagogue effect of NPK was blocked by both aCRH and the ACTH receptor antagonist corticotropin-inhibiting peptide, thereby suggesting that it may be indirectly mediated by the activation of the intramedullary CRH–ACTH system (17). Our present findings are in keeping with this hypothesis, because they clearly show that both NKA and NPK significantly enhance the release of both CRH and ACTH by rat adrenal medulla. The present finding that NKA is less effective than NPK appears to be in keeping with the hypothesis that NPK is the endogenous biologically active tachykinin, NKA being a sequential degradation product (10): an hypothesis that agrees with the lack of effect of NKA on hypophyseal ACTH release (8,22). The possibility that NKA and NPK stimulate the corelease of CRH and ACTH can be easily ruled out, inasmuch as a-CRH blocks the effect of both tachykinins on ACTH secretion, without
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FIG. 3. Effect of -CRH (10–6 M) on basal and NKA (10 06 M)- or NPK (10 06 M )-stimulated ACTH release by rat adrenal medulla tissue. Values are means SE (n Å 6). *p õ 0.01 vs. the respective basal value (B); A p õ 0.01 vs. the respective control group.
FIG. 4. Lack of effect of a-CRH (10 06 M ) on basal and NKA (10 06 M)- or NPK (10 06 M)-stimulated CRH release by rat adrenal medulla tissue. Values are means SE (n Å 6): * p õ 0.01 vs. the respective basal value (B).
affecting that of CRH. The possibility of a toxic nonspecific effect of a-CRH can be excluded, because this antagonist does not alter the basal release of the two peptides. Hence, it seems reasonable to conceive that the primary action of NKA and NPK is the stimulation of the release by chromaffin cells of CRH, which in turn evokes the local ACTH secretion in a paracrine or autocrine manner. This hypothesis accords well with the demonstration that adrenal chromaffin cells are provided with CRH receptors (1,7,24). The presence of tachykinin immunoreactivities in the adrenal medulla of several mammalian species, including the rat, has been demonstrated by both RIA (4–6,22,26) and immunocytochemistry (11,27). Therefore, in light of our present findings, NKA and NPK may be included in that group of intra-adrenal regulatory peptides, which activate intramedullary CRH–ACTH system, thereby indirectly stimulating glucocorticoid secretion of the cortex. At present, this group appears to include argininevasopressin, pituitary adenylate cyclase-activating polypeptide, neuromedin U8, and interleukin-1 [reviewed in (20)].
NKB and SP seem to be almost completely ineffective on the intramedullary CRH–ACTH system, which suggests that the NK2 is the receptor subtype mediating this effect of tachykinins (see Introduction). The very weak stimulatory action of NKB on CRH, but not ACTH, release by adrenal medulla remains to be investigated. It may be tentatively suggested that the effect of this tachykinin is too slight to determine a local concentration of CRH sufficient to induce a sizable ACTH release. In this connection, it is to be recalled that NKB, when systemically administered, appears to induce very doubtful effects on the pituitary ACTH release (14). The lacking of any appreciable effect of SP on the CRH and ACTH release by rat adrenal chromaffin cells agrees with the demonstration that this peptide does not affect either pituitary ACTH or glucocorticoid secretion in rats (16,18). SP has been reported to inhibit hypothalamic CRH secretion (8), a finding that could suggest this may also occur in the adrenal medulla. However, it is conceivable that, due to the very low basal rate of CRH release by chromaffin cells, the inhibitory action of SP cannot be able to elicits detectable effects.
REFERENCES 1. Aguilera, G.; Millan, M. A.; Hauger, R. L.; Catt, K. J. Corticotropinreleasing factor receptors: Distribution and regulation in brain, pituitary and peripheral tissues. Ann. NY Acad. Sci. 512:48–66; 1987. 2. Andreis, P. G.; Neri, G.; Mazzocchi, G.; Musajo, F. G.; Nussdorfer, G. G. Direct secretagogue effect of corticotropin-releasing factor on the rat adrenal cortex: The involvement of the zona medullaris. Endocrinology 131:69–72; 1992.
3. Bagdy, G.; Calogero, A. E.; Szemeredi, K.; Chrousos, G. P.; Gold, P. W. Effects of cortisol treatment on brain and adrenal corticotropin-releasing hormone (CRH) content and other parameters regulated by CRH. Regul. Pept. 31:83–92; 1990. 4. Basile, S.; Cheung, N. S.; Livett, B. G. Chromatographic evidence for the presence of multiple tachykinins in the bovine adrenal medulla J. Neurochem. 58:1584–1586; 1992.
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