Preliminary Study Of Morphological Features And The Possible Role In Mediation No Neurotransmitter Signal Transduction Of Interstitial Cells Of Cajal In The Guinea Pig Corpus Cavernosum

Background and Objective: In1893, Spanish neuroanatomist Santiago RamónyCajal observed a special class of non-neural and neural-related interstitial cells in thegastrointestinal tract (GT) nervous system by methylene blue and silver staining method.The cells were described in detail in the report. Because located in the range between thenerve endings and smooth muscle, these special cells were named for interstitial cells ofCajal (ICC). In the following more than one century, accompanied by the rapiddevelopment of experimental techniques, the researchof the gastrointestinal tract ICC wasdeepening. Now the think of gastrointestinal ICC main functions include:â‘ ICC aspacemaker cells in the gastrointestinal tract, generate the spontaneous rhythm of slow wave(SW) potential, and conduct synchronously to the surrounding smooth muscle cells (SMC)by the ICC intercellular network structure, and result in activating voltage-dependent Ca²⁺channels and generating action potential (AP) in SMC;â‘¡the GT ICC are located betweennerve and SMC, mediate neurotransmitter signaling conduction to the SMC, and participatein the the neural regulation of the gastrointestinal motility. At present, it has been foundthat the ICC response to acetyl choline (ACh), nitric oxide (NO), vascular activity ofintestinal peptide (VIP), adenosine triphosphate (ATP), substance P (SP)and other intestinalneurotransmitters;â‘¢Other functional roles of the GT ICC, such as immune regulation,growth, repair, fibrosis, and participation in the process of some gastrointestinal diseasespathological changes.NO is an important inhibitory neurotransmitter regulating gastrointestinal motility,which is synthesized by nitric oxide synthase (nitric oxide synthase, NOS) catalyticL-arginine in the autonomous nerve endings, because it has a free radical, it can rapidlydiffuse into the target cells. Once the NO neurotransmitter binds with two subunits sGCα,sGCβ of soluble guanylate cyclase (sGC) in the GT SMC, which can subsequently catalyze 3,5-guanosine triphosphate to form cyclic guanosine3′,5′-monophospate (cGMP), thelatter activates cGMP-dependent protein kinases (PKG), the activated PKG phosphorylatestarget proteins, including ion channels, ion pumps and enzymes involved in the regulationof intracellular Ca²⁺levels, and reduces the concentration of intracellular Ca²⁺in the SMC,inhibites excition of the gastrointestinal SMC, results in the relaxation effect of thegastrointestinal smooth muscle.Previous studies have found that the GT ICC, as an intermediator, play an importantrole in mediating NO neurotransmitter signaling to SMC. Nelson G firstly reported that thecolonic ICC can enlarge the relaxation effect of NO neurotransmitter to the smooth muscle.The authors believed that NO neurotransmitter reacted on the intramuscular ICC (ICC-IM),elevated intracellular free Ca²⁺concentration ([Ca²⁺]i) of ICC-IM, which subsequently ledto the further release of NO and the further increase of [Ca²⁺]i, this positive feedback effectamplified nerve neurotransmitter NO inhibitory effect on the GT smooth muscle. Recentstudies have provided a large number of morphological and functional evidences to confirmthe important roles of the GT ICC involved in the mediation of gastrointestinal NO/cGMPsignaling pathway. These evidences include:â‘ TheGT ICC expressed more sGC than theSMC, which was the sensitive receptor of the NO neurotransmitter, the result indicated thatthe GT ICC were the primary target cells of NO;â‘¡The functional immunohistochemistryresults showed that neural-release NO as well as exogenous NO cound lead to the increaseof cGMP in the GT ICC, while similar effect of the GT SMC to exogenous NO was notobvious;â‘¢In the c-kit mutation W/WVrats, the relaxant effect of NO neurotransmitter toisolated smooth muscle was significantly diminished;â‘£The expression of nNOSon theGT ICC might be associated with the signal conversion of ICC-mediated neural signalingprocess;⑤Theclose contact, which was similar to synapses, was formed between the GTICC and nearby nNOS-Immunoreactive nerve endings, but it was not found between the GTSMC and the autonomic nervous system. We generalize the current studies of the GT ICC,there may be two ways to participate in the transmission mechanism of NOneurotransmitter signaling:â‘ NO neurotransmitteris synthesized and release from theautonomous nerve endings, react with sGC in the nearby ICC through the contact of similarsynapsis, actived sGC lead to synthesize a lot of cGMP in in the cytoplasm, which cansubsequently activate PKG, then leads to cellular responses, electrophysiological effects can transmit to the SMC by the gap junction between the ICC and SMC, result in the latterhyperpolarization steady-state membrane potential, weaken the contraction of smoothmuscle cells to excited neurotransmitter or lead to relaxation of smooth muscle;â‘¡theGTICC express the nNOS, when the NO neurotransmitter react with the receptor of ICC,may be caused ICC intracellular Ca²⁺concentration will be increased and NO or its analogssynthesis will be released by some mechanisms, in order to enlarge the neurotransmitter NOinhibitory effect on SMC.In the penis erectile mechanism, the NO/cGMP/PKG signaling pathway is the mostimportant mechanism to regulate the corpus cavernosum smooth muscle relaxation andpenile erection. The traditional view is that non-cholinergic non-adrenergic (NANC) nerveendings and endothelial cells can synthesize NO molecule, in the corpus cavernosum, NOact directly on the SMC by NO/cGMP/PKG signaling pathway, which results inrelaxation of smooth muscle and penile erection effect. But the mechanism of NOneurotransmitter-induced penile erection is not fully explained clearly, it is not knownwhether there are orther NO signaling pathways, except for the direct mode of NO actingon SMC.The c-kit immunoreactive ICC have been indentified in the Corpus cavernosum tissueof human, guinea pigs and other species. Morphological and electrophysiological studieshave confirmed the cavernous ICC is not the pacemaker cells, its main roles may serve asintermediary to mediate neurotransmitter signaling to regulate the motion of cavernoussmooth muscle. Previous studies have found that the spontaneous contraction of corpuscavernosum smooth muscle can be inhibited or weakened respectively by indomethacin orNS398(COX-2cyclooxygenase inhibitors), cavernous ICC on COX-2immune stronglypositive, but the SMC on COX-2immunoreactive very weak, suggesting that the thecavernous ICC as an intermediary may be involved in the in vivo release of prostaglandinregulation of spontaneous contraction of cavernous smooth muscle. Pathological study alsofound that the cavernous tissue of patients with erectile dysfunction (ED), the cells numberof ICC significantly reduced, so to speculate that this may be related to the incidence of EDIn summary, the research of the corpus cavernous ICC is still in the initial stage, basedon the roles of the GT ICC in mediating NO neurotransmitter signaling transmission and aswell the research findings of penis ICC, this project intends to explore the penis ICC may be involved in NO-mediated neurotransmitter to regulate smooth muscle relaxation effectfrom two aspects of the morphology and function, it is summarized as the NOneurotransmitter-> ICC-> SMC signal transduction pathways. This project will provide anew entry point to further research of the physiological mechanism of penile erection andpathogenesis studies of ED.Materials and methods:1.6-8month-old healthy male guinea pigs were used in the study, the features ofmorphology and distribution of the cavernous ICC were observed throughimmunohistochemistry experiment of corpus cavernosum;2. Ultrastructural features and the relationship between the cavernous ICC and theperipheral nerves or the nearby SMC were observed by transmission electron microscopyexperiments, in order to provide morphological evidence for the functional research of theICC;3. The method of sorting ICC cells that can be used for molecular biology researchwas established by flow cytometry technology, and prepare for the follow-up ICCfunctional investigation;4. The expression of NO receptor protein on the penis ICC was examined by theWestern Bloting and immunofluorescence histochemistry, in order to verify that thecavernous ICC were target cells of the NO neurotransmitter;5. The expression of the NO neurotransmitter signaling downstream molecules PKG,nNOS on the penis ICC was detected by immunofluorescence histochemistry experiments,in order to discuss the possible mechanisms how the cavernous ICC mediated the NOneurotransmitter signaling transmission to regulate relaxation effect of the smooth muscle.Results:1. Immunohistochemical experiment of adult male guinea pig corpus cavernosumtissue slices staining with the c-kit antibody showed that typical c-kit immunopositive ICCpresented in guinea pig corpus cavernosum, they were characterized by a fusiform cell body,with dendritic processes at each pole (bipolar) or alongthe sides of the cell body(multipolar). They were about the size of40nm-110nm (63.7±24.2nm). The cavernousICC were located among the SMC or in the subendothelial tissue of blood vessels andcavernous. They were lack of mutual contact and can not form the cellular network; 2. TEM revealed the ultrastructural features of the corporal ICC and relationshipsbetween ICC and adjacent SMC and nerves. Lower-magnification TEM images showed thatICC had a narrow perinuclear cytoplasm and an oval nucleus, they are often located alongthe boundary of smooth muscle bundles and were close tointramural nerve fibers.Higher-magnification micrographies suggested the ultrastructure of ICC in the guinea pigcorporal tissue, which were characterized by abundant caveolae along the cellmembrane,numerous mitochondria and densebodies, a few of rough endoplasmic reticulumandlysosomes in the cytoplasm. Intermediate filaments were not prominent. Gap junctionswere clearly observed between the corporal ICC and the SMC.3. The method of sorting ICC cells that can be used for molecular biology researchwas established successfully by flow cytometry technology;4．Western Bloting and immunofluorescence histochemistry experiments confirmedtwo subunits sGCα1, sGCβ1of the NO receptor sGC express on the guinea pig corpuscavernosum ICC;.5. Immunofluorescence histochemical experiments confirmed the expression of theNO neurotransmitter signaling downstream molecules PKG, nNOS on the penis ICC.Conclusion:1. morphologic study of guinea pig corpus cavernosum ICC found that theirmorphology, distribution, ultrastructural features and the relationships between ICC and thesurrounding SMC and nerve tissue. The cavernous ICC were characterized by a fusiformcell body, with dendritic processes at each pole (bipolar) or alongthe sides of the cell body(multipolar). They were located among the SMC or in the subendothelial tissue. They werelack of mutual contact and the cellular network. ultrastructural features of the cavernousICC were observed by TEM, which are similar to the GT ICC. In addition, the ICC werefound close to intramural nerve fibers, and extensive gap junctions were shown betweenICC and adjacent SMC. Morphologic study provides the structural basis for the functionalinvestigation of the cavernous ICC.2. The cavernous ICC that can be used for molecularbiologic research were isolatedand purified successfully by flow cytometry technology. A simple, viable cell-sortingmethod is established for the following experiments.3. Functional investigations of guinea pig corpus cavernosum ICC found expression of sGC, sensitive receptor protein of the NO neurotransmitter, and nNOS, the PKG proteinon the cavernous ICC. The results suggest that the cavernous ICC were target cells of NOneurotransmitter, when NO neurotransmitter released from nitrergic neural system act onthe intracavernous ICC, which may play similar roles to the ICC of the gastrointestinal tract,NO was further synthesized and released from the cavernous ICC and responded to thesurrounding SMC, or activated the PKG in the cavernous ICC, led to potential changes ofICC, propagated the electrical signal to the adjacent SMC through the gap junction andmodulated relaxation of the cavernous smooth muscle.