Investigation Of Corticotrophin-Releasing Factor Family Peptides On Voltage-Gated Calcium Channels

BACKGROUNDCorticotropin-releasing factor (CRF) belongs to a family of structurally relatedpeptides that includes fish urotensin 1, amphibian sauvagine, and the recentlyidentified urotensin homologue, urocortins, which includes urocortin 1-3. CRF familyhas two receptors, CRFR1 and CRFR2. Urotensin 1, sauvagine, and urocortin 1-3bind with up to 1000-fold higher affinities to the CRFR2 than species homologues ofCRF. UCN2 and UCN3 are generally considered to represent endogenous ligands formammalian CRFR2 variants, whereas UCN is thought to be an endogenous ligand forboth CRFR1 and CRFR2. UCN shares 43ï¼…homology with CRF at the amino acidlevel. Previous reports showed that UCN was involved in the modulation of a varietyof biological activities, such as causing a dose-dependent increase in coronary,testicular artery blood flow suppression of appetite, and maintenance of the placentalfunction & labor. CRF was observed to have modulating effects on male reproductionearly in late 80's. It was reported that UCN could activate the myometrial contractility.In addition, presence of UCN in the seminal fluid could be of relevance duringfertilization and pregnancy by influencing sperm transport through the female genitaltract. Therefore, these findings suggest that the CRF family, including UCN, CRF, andits receptors, may play important roles in various biological functions during the course of reproduction, which is highly associated with calcium channels. Previousreports also have showed that urocortin 2 had a number of physiological properties byinteracting with its CRFR2. However, urocortin 2 could also cause neural activationin cell groups that are involved in autonomic activation, but do not express either typeof CRF receptor. However, to date, the exact mechanisms for UCNs' effects viaCRFR-dependent or independent manner are not very clear. It has been demonstratedthat UCNs, espcically UCN and UCN2 protected neonatal cardiac myocytes in vitrowhen administered before hypoxia/ischemia, which suggests that calcium channelsmay play some roles since ischemia and hypoxia damage is highly associated withCa²⁺-overload. Furthermore, it was reported that UCN2 played the cardiovascularprotective role via protein kinase A (PKA), PKC, PLC, and mitogen-activated proteinkinase (MAPK) pathway, which might lead to changes in the intracellular Ca²⁺, anddownstream regulation of the calcium channels.Voltage-gated calcium channels (VGCCs) first described in excitable cells aregenerally classified according to their electrophysiological, pharmacological andinactivation properties as T-type, long-lasting L-type, neuronal N-type, purkinje cellP-type, granular cell Q-type and toxin/drug-resistant R-type. So far, however, fewreports addressed UCNs' effects on VGCC. In addition, whether the effecs are CRFreceptor dependent or independent is still unknown.PURPOSE1. The aim of this study was to investigate the expression of UCN & its receptors and the effects of UCN on male reproductive functions, and identify the mechanismsof these effects relevant to intracellular calcium.2. The second goal is to investigate the effects of UCN on T-type calcium channels,and explore the mechanisms of UCN's role in male reproductive functions,especially in acrosome reaction (AR) and sperm motility.3. Thirdly, we aim to clarify the subcellular signaling pathway between CRFR1 andthe three isoforms of T-type calcium channels, and explore the mechanisms forneuroendrine systems.4. Another goal of this study is to observe the expressions of UCN2 in neural system,and the ion mechnisms for UCN2's neuroprotective effects.5. The last aim of this study is to explore the vascular protection of UCN2, charify theinhibition of UCN2 on vascular remodeling, and investigate the ion mechanismsrelevant to L-type calcium channels.METHODS1. Separate locations of UCN and its receptors in mouse testis and functions of UCNin male reproduction and the relevant mechanisms: Preparation of spermatozoaand spermatogenic cells, Reverse transcription-polymerase chain reaction(RT-PCR), Immunohistochemical staining, Double immunostaining for UCN,Measurement of sperm motility, Assay for acrosome reaction (AR), Determinationof [Ca²⁺]_i by flow cytometry2. Mechanisms of UCN's effects on male reproductive in mice: Spermatogenic cell preparation and electrophysiology3. Activation of CRFR1 selectively inhibits Ca_v3.2 calcium channels: RT-PCR,Western, and Confocal Microscopy detect the expression of CRFR1 in HEK293,cAMP detection, Whole-cell patch clamp, Pharmacological methods (PKA, PKC,PLC, CaMK, QEHA, and SKEE etc.), Construct rat CRFR14. Expression of UCN2 and mechanisms of its neuroprotection: Cell culture forrat undifferentiated pheochromocytoma (PC12) cells, Acute isolation of cerebralcortex neurons, Reverse transcription-polymerase chain reaction (RT-PCR),Measurements of [Ca²⁺]_i by confocal laser scanning microscopy, Whole-CellPatch Clamp Recording5. Effects of UCN2 on vascular remodeling and the relevant mechanisms: Isolationof mesenteric arteries smooth muscle cells (MASMC) from SHR, TUNEL assayto detect the apoptosis of VSMC in SHR, Measurement of nitrite oxide (NO),Intracellular Ca²⁺ concentration ([Ca²⁺]_i) measurements, Transient expression ofhuman Cav1.2 in HEK293 Cells, Whole-cell patch clamp recordingRESULTS1. UCN expressed only in mature sperm while CRFR1 and CRFR2 existed inspermatocytes and spermatogonia, respectively. With AR proceeding, theexpression of UCN declined, and after ascrosome reaction, UCN could not bedetected any more.2. UCN significantly inhibited the motility of the sperm and AR in a concentration-dependent manner.3. UCN reduced the [Ca²⁺]_i increase induced by high potassium and T-type calciumcurrents.4. Activation of CRFR1 selectively inhibited Cav3.2, instead of Cav3.1 and Cav3.3.5. HEK293 cells endogenously expressed functional CRFR1. The CRFR1 waslocalized in cell membrane, and activated the increase of cAMP.6. CRFR1 activation affected Cav3.2 channels by negatively shifting steady-stateinactivation properties.7. UCN inhibited Ca_v3.2 T-type calcium channels independent of PKA, PKC andPLC. UCN inhibited Ca_v3.2 T-type calcium channels via G_βγ.8. UCN2 reduced the [Ca²⁺]_i levels via inhibiting L-type VGCC in undifferentiatedPC12 cells, where both UCN2 and CRFR2β, but not CRFR1 or CRFR2α, wereexpressed.9. UCN2 reduced [Ca²⁺]_i, inhibited MASMC apoptosis induced by hypoxia in SHR,and blocked Cav1.2.CONCLUSIONS1. UCN and its recepotrs endogenously express in mouse testis. UCN inhibits themale reproductive functions.2. Activation of CRFR1 inhibits T-type calcium channels, which may be responsiblefor its male reproductive inhibition.3. Activation of CRFR1 inhibits Ca_v3.2 T-type calcium channels via G_βγ subunits. 4. HEK 293 cells endogenously express functional CRFR1, which may be used as anew transfection model for CRFR1.5. We provides evidence that UCN2, co-expressed with CRFR2βin undifferentiatedPC12 cells, decreases [Ca²⁺]_i levels via inhibiting the L-type VGCC. It may be apotential endogenous protective agent under some pathophysiological conditions,such as brain ischemia and some neuronal degenerative diseases like Alzheimer'sand Parkinson's diseases, in which Ca²⁺-overload plays very important roles.6. Our results provide convincing evidence of a possible link between theapoptosis-inhibitory effects of UCN2 and L-type calcium channels in SHR.Based on the theory that VSMC apoptosis plays a key role in hypertensivevasculopathy, these findings highly imply that UCN2 might be a beneficial agentin vascular pathology where Ca²⁺-overload plays an important role.