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.

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 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. P<0.05 was considered statistically significant.

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 30^st and 60^st minutes after treatment, but it was not statistically significant (Figure 1).

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 0^th minute in 3pmol ghrelin administered group (p<0.05 and p<0.01, respectively, Figure 2).

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.

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.