Serotonin in trigeminal ganglia of female rodents: relevance to menstrual migraine.

OBJECTIVES: We examined changes in the serotonin system across the estrous cycle in trigeminal ganglia of female rodents to determine which components are present and which are regulated by the variations in levels of ovarian steroids that occur during the estrous cycle. BACKGROUND: Migraine is 2-3 times more prevalent in women than in men and attacks are often timed with the menstrual cycle, suggesting a mechanistic link with ovarian steroids. Serotonin has been implicated in the pathogenesis of migraine, and the effectiveness of triptans, selective 5HT-1B/D/F agonists, has provided further support for this concept. It is not known whether serotonin, its rate-limiting enzyme tryptophan hydroxylase (TPH), or its receptors are regulated by ovarian steroids in trigeminal ganglia. METHODS: We used reverse transcription-polymerase chain reaction to examine gene expression in cycling mice, Western blots to examine protein expression, double-labeling immunohistochemistry using markers of nociceptors and nonnociceptors and confocal microscopy to identify specific types of neurons, and primary tissue culture to examine effects of estrogen on trigeminal neurons in vitro. RESULTS: In C57/BL6 mice mRNA levels of TPH-1, the rate-limiting enzyme in serotonin synthesis, were over 2-fold higher and protein levels were 1.4-fold higher at proestrus, the high estrogen stage of the cycle than at diestrus, the low estrogen stage. TPH protein also was present in primary trigeminal cultures obtained from female Sprague-Dawley rats, but levels were not affected by 24-hour treatment with physiological levels (10(-9) M) of 17beta-estradiol. Gene expression of 5HT-1B and 5HT-1D receptors in trigeminal ganglia was not regulated by the estrous cycle. Serotonin was present in trigeminal neurons containing CGRP, a potent vasoactive neuropeptide, peripherin, an intermediate filament present in neurons with unmyelinated axons, neurofilament H, which is present in neurons with myelinated axons, and in neurons binding IB4, a marker of nonpeptidergic nociceptors. Serotonin was also present in neurons containing 5HT-1B. The serotonin-positive population was significantly larger in diameter than the serotonin-negative population. Conclusions.-Expression of the rate-limiting enzyme required for serotonin synthesis is regulated during the natural estrous cycle, and serotonin is present in larger trigeminal neurons of all the major subtypes. Colocalization of serotonin with 5HT-1B suggests that this receptor functions as an autoreceptor to regulate serotonin release. Cyclical changes in serotonin levels in trigeminal ganglia could contribute to the pathogenesis of menstrual migraine.

cAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation.

In the regulation of steroid biosynthesis, a process mediated by the steroidogenic acute regulatory (StAR) protein, both cAMP-dependent and -independent pathways are involved. While the cAMP-dependent regulatory events represent, by far, the most robust increase in steroid synthesis and are well established, the knowledge regarding cAMP-independent mechanisms is lacking. The present investigation was designed to elucidate the potential involvement of the latter in regulating StAR expression and steroidogenesis in mouse Leydig tumor cells (mLTC-1 cells). Treatment of mLTC-1 cells with a number of factors including insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor, transforming growth factor (TGF)alpha, interleukin-1 (IL-1), and colony-stimulating factor-1, increased the levels of StAR mRNA, StAR protein, and progesterone to varying degrees and utilized signaling pathways that are not associated with elevations in intracellular cAMP levels. Importantly, phosphorylation of StAR in response to these stimuli was undetectable, which is in marked contrast to observations with human chorionic gonadotropin (hCG), indicating factors that do not alter intracellular cAMP, regulate the steroid biosynthesis in a StAR phosphorylation-independent manner. In addition, the roles for factors involved in cross-talk between the protein kinase pathways, PKA and PKC, were demonstrated. Further characterization of signaling by one such cAMP-independent factor, TGFalpha, demonstrated that the mechanism, whereby it increased StAR expression and steroid synthesis, was dependent on de novo protein synthesis and mediated via activation of the EGF receptor. TGFalpha was also able to augment hCG-stimulated cAMP synthesis, StAR protein and StAR phosphorylation, and influence hCG binding and LH receptor mRNA expression. Furthermore, TGFalpha increased phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and cAMP-response element-binding protein (CREB), processes inhibited by the mitogen-activated protein kinase/ERK inhibitor U0126 and by expression of non-phosphorylatable CREB-M1 respectively. Inhibition of ERK activity enhanced TGFalpha-mediated StAR protein expression (but not its phosphorylation) and decreased progesterone synthesis, events correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). Collectively, these findings demonstrate that, in mouse Leydig cells, cAMP-independent signaling events regulate steroidogenesis in a StAR phosphorylation-independent manner.

Molecular mechanisms of insulin-like growth factor-I mediated regulation of the steroidogenic acute regulatory protein in mouse leydig cells.

Growth factors are known to play diverse roles in steroidogenesis, a process regulated by the mitochondrial steroidogenic acute regulatory (StAR) protein. The mechanism of action of one such growth factor, IGF-I, was investigated in mouse Leydig tumor (mLTC-1) cells to determine its potential role in the regulation of StAR expression. mLTC-1 cells treated with IGF-I demonstrated temporal and concentration-dependent increases in StAR expression and steroid synthesis. However, IGF-I had no effect on cytochrome P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase protein levels. IGF-I was capable of augmenting N,O'-dibutyrl-cAMP-stimulated steroidogenic responsiveness in these cells. The steroidogenic potential of IGF-I was also confirmed in primary cultures of isolated mouse Leydig cells. IGF-I increased phosphorylation of ERK1/2, an event inhibited by the MAPK/ERK inhibitors, PD98059 and U0126. Interestingly, inhibition of ERK activity enhanced IGF-I-mediated StAR protein expression, but phosphorylation of StAR was undetectable, an observation in contrast to that seen with N,O'-dibutyrl-cAMP signaling. Further studies demonstrated that these events were tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 and scavenger receptor class B type 1. Whereas both protein kinase A and protein kinase C signaling were involved in the IGF-I-mediated steroidogenic response, the majority of the effects of IGF-I were found to be mediated by the protein kinase C pathway. Transcriptional activation of the StAR gene by IGF-I was influenced by several transcription factors, its up-regulation being dependent on phosphorylation of the cAMP response element-binding protein (CREB) and the activator protein 1 family member, c-Jun. Conversely, StAR gene transcription was markedly inhibited by expression of nonphosphorylatable CREB (Ser(133)Ala), dominant negative A-CREB, and dominant negative c-Jun (TAM-67) mutants. Collectively, the present studies identify molecular events in IGF-I signaling that may influence testicular growth, development, and the Leydig cell steroidogenic machinery through autocrine/paracrine regulation.

Ghrelin is expressed in trigeminal neurons of female mice in phase with the estrous cycle.

Several disorders mediated by the trigeminal nerve including migraine and temporomandibular disorder (TMD) are more common in women than in men, and painful attacks are often linked to the menstrual cycle. Estrogen receptors in trigeminal neurons may be involved in regulating neuronal function, causing changes in sensitivity that contribute to these attacks. In a previous study, we demonstrated that expression of specific neuropeptides including galanin and neuropeptide Y in trigeminal ganglia of female rodents varies with the estrous cycle. In this study, we examined expression of the orexigenic peptide ghrelin in trigeminal ganglia of cycling female mice. RT-PCR studies demonstrated that ghrelin mRNA is upregulated by over 5-fold at the high estrogen stages of the cycle, proestrus and early estrus over the levels expressed at the low estrogen stage of the cycle, diestrus. Double-labeling immunohistochemical studies and cell size measurements were conducted to identify the phenotype of neurons in trigeminal ganglia containing ghrelin. Ghrelin was present in trigeminal neurons containing peripherin, a marker of neurons with unmyelinated axons, in trigeminal neurons binding IB4, a marker of nonpeptidergic nociceptors, in trigeminal neurons containing neurofilament H, a marker of neurons with myelinated axons, and in trigeminal neurons containing the neuropeptide calcitonin gene-related peptide (CGRP). Ghrelin-positive neurons averaged 25.6 microm in diameter, but included neurons in all the size ranges except the smallest peripherin-positive neurons. Thus, nearly all of the major populations of trigeminal neurons including peptidergic and nonpeptidergic nociceptors contain ghrelin. These studies suggest that ghrelin, a multifunctional peptide, may contribute to the mechanism linking orofacial pain syndromes in females, including temporomandibular disorder and migraine, to cyclical hormonal changes.