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Fırat Tıp Dergisi
2005, Cilt 10, Sayı 3, Sayfa(lar) 089-091
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Effect of Ghrelin on Pain Threshold in Mice
Selim KUTLU1, Mete OZCAN2, Sinan CANPOLAT1, Süleyman SANDAL1, Mehmet AYDIN1, Haluk KELESTİMUR1
1Fırat Üniversitesi, Tıp Fakültesi, Fizyoloji, Anabilim Dalı, ELAZIĞ
2Fırat Üniversitesi, Tıp Fakültesi, Biyofizik, Anabilim Dalı ,ELAZIĞ
Keywords: Grelin, ağrı eşiği, hot plate ve fare, Ghrelin, pain threshold, hot plate and mice
Summary
Objectives: Ghrelin, a novel growth hormone-releasing peptide, was isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogues receptor. The present study was planned to determine whether ghrelin affects pain threshold in mice.

Materials and Methods: Adult male BALB/C mice weighing 25–30g were used in this study. The hot plate test was conducted by placing the mouse on a metal surface maintained at 50±0.1ºC by using hot plate analgesia meter. The latency to jumping or licking a hind paw was recorded as nociceptive threshold. Animals were allowed to acclimate to the hot plate for a period of 1 week prior to the experiment. Different doses of ghrelin were intraperitoneally administered to the animals after control latencies. Control group received saline alone. Hot plate test were performed in all animals individually in 30 th, 60 th, 90 th and 120 th minutes after injection. Pain threshold values were determined and analyzed by Mann-Whitney U Test and Wilkoxon Sign Ranks Test.

Results: Ghrelin didn’t affect pain threshold throughout the experiment in 0.3pmol and 1pmol doses compared to control values. There were significant decreases in pain threshold when it is given in a dose of 3pmol in 30 th and 60 th minutes (p<0.05 and p<0.01, respectively).

Conclusion: The results of this study have presented that ghrelin may have a decreasing effect on pain threshold in mice. Further studies are needed to determine the mechanism by which ghrelin exerts its nociceptive effect. ©2005, Fırat University, Medical School

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  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Introduction
    Ghrelin, an acylated 28-amino acid peptide recently isolated from mammalian stomach, has been identified as an endogenous ligand for growth hormone secretagogue receptors 1. This hormone exerts a strong stimulatory effect on GH secretion in humans 2 and rats 3. Ghrelin stimulates food intake after central and peripheral administration 4. Food intake causes a decrease in ghrelin level in blood 5. In addition to these effects, this hormone may be involved in some physiological processes in the central nervous system. In the brain, receptors for ghrelin have been detected in multiple hypothalamic nuclei as well as in the hippocampus, substantia nigra, ventral tegmental area, and dorsal and median raphe nuclei 6;7. Ghrelin acts at the nucleus of the solitary tract to suppress sympathetic activity and to decrease arterial pressure in rats 8. Ghrelin increase anxiety-like behavior and memory retention 9. It is suggested that ghrelin is an endogenous sleep-promoting factor in humans 10.

    It has been shown that obese people or animals may have different responses to pain stimuli. The sensory and pain threshold were found to be higher in the obese people than in the control subjects. The patients with fatness had higher pain sensitivity threshold than people of other categories, so they felt less pain. Dietary-induced obese rats were found to be similar to obese humans in being less sensitive to painful stimuli. Ghrelin level is reduced in obese human 11 and rodents 12. Therefore, it may be thought that there is a relationship between ghrelin and nociception. In this study we investigated the possible effect of ghrelin on pain threshold in mice.

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  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Methods
    Adult male Balb/C mice weighing 25-30g obtained from Firat University Biomedical Unit (Elazig) were used in this study (n=36). They were housed under controlled light (12-h light and 12-h dark, lights on at 07.00h) and temperature (21±1ºC) conditions. Food and water were supplied ad libitum.

    The hot plate test was conducted by placing the mouse on a metal surface maintained at 50±0.1ºC using the hot plate analgesia meter (Harward Apparatus Ltd., England). Hot plate was surrounded with a transparent plastic barrier. The latency to jumping or licking a hind paw was recorded. In the absence of a response, the animal was removed 60s after the placement into the hot plate to prevent tissue damage. Animals were allowed to acclimate to the hot plate for a period of 1 week prior to the experiment.

    Different doses of ghrelin (0.3pmol (n=10), 1pmol (n=8) and 3pmol (n=8)) were intraperitoneally administered to the animals after obtaining control latencies (minute 0). Control group received saline alone (n=10). Hot plate test was performed on all animals individually in 30th, 60 th, 90 th and 120th minutes after injection. Pain threshold values were determined and analyzed by Mann-Whitney U Test and Wilkoxon Sign Ranks Test. P<0.05 was considered statistically significant.

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  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Results
    The response latencies had similar values throughout the experiment in the control group. 0,3 pmol ghrelin did not have any significant effect compared to control group (Figure 1). The second dose of ghrelin (1pmol) slightly decreased the pain threshold in 30st and 60st minutes after treatment, but it was not statistically significant (Figure 1).


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    Figure 1: Hot plate latencies (Mean±SEM) of control (n=10,), 0,3 pmol (n=10), 1 pmol (n=8) and 3 pmol (n=8) ghrelin administered groups. *p<0.05 and **p<0.01 compared to control by using Mann-Whitney U Test

    There were significant decreases in pain threshold when ghrelin was administered at a dose of 3pmol in 30 th and 60 th minutes compared to control group (p<0.05 and p<0.01, respectively, Figure 1). Additionally, significant decreases were occurred in 30 th and 60 th minutes compared to beginning value in 0th minute in 3pmol ghrelin administered group (p<0.05 and p<0.01, respectively, Figure 2).


    Click Here to Zoom
    Figure 2: Hot plate latencies (Mean±SEM) of 3 pmol ghrelin. *p<0.05 and **p<0.01 compared to control (0th minute) by using Wilkoxon Sign Ranks Test

    We also observed that mice started food intake at 15 minutes after injection of 3pmol ghrelin. But the aim of this study was not to determine the effect of ghrelin on food intake, so we ignored the feeding behavior of animals.

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  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Discussion
    The results of the present study demonstrate that ghrelin has hiperalgesic effect in mice. This is only a preliminary study and no attempt was made to clarify the possible mechanism of action of ghrelin on pain threshold. This is the one possibility that ghrelin may induce the Ca2+ entry into the neuronal tract involved in transporting noxious stimuli. There is not any direct evidence about this insight. However, ghrelin induces an increase in the intracellular calcium concentration in porcine somatotropes via L-type calcium channel in a dose dependent manner 13. In a zero Na+ solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channel, sodium channels are also involved in ghrelininduced calcium transients 13. Additionally, ghrelin directly interacts with NPY neurons in the arcuate nucleus to induce Ca2+ signalling via protein kinase-A and N-type calcium channel-dependent mechanisms in rats 14.

    Histaminergic neurons in the tuberomamillary nucleus are implicated in nociception 15 and presence of GHS-R in the tuberomammillary nucleus was suggested by the existence of GHSR mRNA in this area 7. Therefore ghrelin may show its nociceptive effect indirectly by affecting histaminergic transmission. In an electrophysiologic study, it is demonstrated that ghrelin activates histaminergic neurons in the tuberomammillary nucleus by inhibiting G protein-coupled inwardly rectifier K+ channels 16. Injection of histamine into the rat dorsal raphe nucleus and periaqueductal grey region produces an antinociception, while its injection into the median raphe nucleus causes hyperalgesia 17,18. Intracerebroventricular administrations of low doses of histamine elicit hyperalgesia, while high doses of histamine produce antinociception 19,20. The results of above studies suggest that the opposite effects of histamine on pain threshold may be mediated through different subtypes of histamine receptors 20,21.

    It is presented that serotonergic pathways originating from dorsal raphe nucleus is involved in pain modulation 22. Raphe nucleus is another target for the effect of ghrelin 9. Ghrelin has been found to decrease serotonin release in hypothalamus in vitro 23 and suggested to decrease serotonin release in dorsal raphe nucleus 24, which may have an additional role in food intake-increasing effect of ghrelin. Therefore, ghrelin may also affect analgesic system due to its effects on serotonergic transmission in brain stem, which also modulates pain transmission in medulla spinalis.

    In conclusion, ghrelin may be a candidate for hormonal regulation of pain sensitivity. However, further studies are needed to establish its effect on nociception and the mechanism by which it exerts its effect.

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  • Summary
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • References

    1) Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growthhormone- releasing acylated peptide from stomach. Nature 1999; 402: 656-60.

    2) Arvat E, Di Vito L, Broglio F, et al. Preliminary evidence that Ghrelin, the natural GH secretagogue (GHS)-receptor ligand, strongly stimulates GH secretion in humans. J Endocrinol Invest 2000; 23: 493-5.

    3) Date Y, Murakami N, Kojima M, et al. Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats. Biochem Biophys Res Commun 2000; 275: 477-80.

    4) Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000; 407: 908-13.

    5) Ariyasu H, Takaya K, Tagami T, et al. Stomach is a major source of circulating ghrelin, feeding state determines plasma ghrelinlike immunoreactivity levels in humans. J Clin Endocrinol Metab 2001; 86: 4753-8.

    6) Lu S, Guan JL, Wang QP, et al. Immunocytochemical observation of ghrelin-containing neurons in the rat arcuate nucleus. Neurosci Lett 2001; 321: 157-60.

    7) Guan XM, Yu H, Palyha OC, et al. Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 1997; 48: 23-9.

    8) Lin Y, Matsumura K, Fukuhara M, et al. Ghrelin acts at the nucleus of the solitary tract to decrease arterial pressure in rats. Hypertension 2004; 43: 977-82.

    9) Carlini VP, Monzon ME, Varas MM, et al. Ghrelin increases anxiety-like behavior and memory retention in rats. Biochem Biophys Res Commun 2002; 299: 739-43.

    10) Weikel JC, Wichniak A, Ising M, et al. Ghrelin promotes slowwave sleep in humans. Am J Physiol Endocrinol Metab 2003; 284: 407-15.

    11) McLaughlin T, Abbasi F, Lamendola C, Frayo RS, Cummings DE. Plasma ghrelin concentrations are decreased in insulinresistant obese adults relative to equally obese insulin-sensitive controls. J Clin Endocrinol Metab 2004; 89: 1630-5.

    12) Ariyasu H, Takaya K, Hosoda H, et al. Delayed short-term secretory regulation of ghrelin in obese animals: evidenced by a specific RIA for the active form of ghrelin. Endocrinol 2002; 143: 3341-50.

    13) Glavaski-Joksimovic A, Jeftinija K, Scanes CG, Anderson LL, Jeftinija S. Stimulatory effect of ghrelin on isolated porcine somatotropes. Neuroendocrinol 2003; 77: 367-79.

    14) Kohno D, Gao HZ, Muroya S, Kikuyama S, Yada T. Ghrelin directly interacts with neuropeptide-Y-containing neurons in the rat arcuate nucleus: Ca2+ signaling via protein kinase A and Ntype channel-dependent mechanisms and cross-talk with leptin and orexin. Diabetes 2003; 52: 948-56.

    15) Schwartz JC, Arrang JM, Garbarg M, Pollard H, Ruat M. Histaminergic transmission in the mammalian brain. Physiol Rev 1991; 71: 1-51

    16) Bajic D, VanManh Hoang Q, Nakajima S, Nakajima Y. Dissociated histaminergic neuron cultures from the tuberomammillary nucleus of rats: culture methods and ghrelin effects. J Neurosci Met 2004; 132: 177-84.

    17) Glick SD, Crane LA. Opiate-like and abstinence-like effects of intracerebral histamine administration in rats. Nature 1978; 273: 547-9.

    18) Thoburn KK, Hough LB, Nalwalk JW, Mischler SA. Histamineinduced modulation of nociceptive responses. Pain 1994; 58: 29- 37.

    19) Chung YH, Miyake H, Kamei C, Tasaka K. Analgesic effect of histamine induced by intracerebral injection into mice. Agents Actions 1984; 15: 137-42.

    20) Malmberg-Aiello P, Lamberti C, Ghelardini C, Giotti A, Bartolini A.Role of histamine in rodent antinociception. Br J Pharmacol 1994; 111: 1269-79.

    21) Lamberti C, Bartolini A, Ghelardini C, Malmberg-Aiello P. Investigation into the role of histamine receptors in rodent antinociception. Pharmacol Biochem Behav 1996; 3: 567-74.

    22) Andersen E, Dafny N. An ascending serotonergic pain modulation pathway from the dorsal raphe nucleus to the parafascicularis nucleus of the thalamus. Brain Res 1983; 269: 57-67.

    23) Brunetti L, Recinella L, Orlando G, et al. Effects of ghrelin and amylin on dopamine, norepinephrine and serotonin release in the hypothalamus. Eur J Pharmacol 2002; 454: 189-92

    24) Carlini VP, Varas MM, Cragnolini AB, et al. Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin. Biochem Biophys Res Commun 2004; 313: 635-41.

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
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