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FUNCIÓN DEL CALCIO EN LA REGULACIÓN VASCULAR PENEANA Y SU IMPLICACIÓN EN LA VASCULOPATÍA DIABÉTICA

NURIA VILLALBA ISABEL

Summary

The contracted state of corpus cavernosum and arterial smooth muscledetermines the maintenance of penile flaccidity, whereas its relaxation allowsthe filling of the sinuses and an initial rise in intracavernosal pressure that leadsto erection (Andersson and Wagner, 1995). Nitric oxide (NO) released fromnonadrenergic-noncholinergic (NANC) nerve endings and endothelial cells uponsexual stimulation is considered to be the main mediator of erection (Anderssonand Wagner, 1995; Simonsen et al., 1997; Prieto et al., 1998; Simonsen et al.,2002). Accordingly, endothelial dysfunction and reduced availability of NO invascular diseases such as hypertension, hypercholesterolemia, and diabetesmellitus are often associated with erectile dysfunction, and enhancement of NOmediatedrelaxations by selective inhibitors of phosphodiesterase 5 constitutesthe basis of current oral treatments successfully used for organic impotence(Moreland et al., 2001). During the flaccid state, erectile tissue is contracted bythe release of neural and local factors, such as norepinephrine, thromboxaneA2, neuropeptide Y and endothelin-1 that increase smooth muscle cytosolicCa2+ ([Ca2+]i) and/or Ca2+ sensitization through activation of G protein-coupledreceptors (Andersson and Wagner, 1995; Simonsen et al., 2002; Prieto et al.,2004). Tonic physiological activity of RhoA/Rho kinase is also involved in theregulation of erectile function and the maintenance of penile flaccidity (Chitaleyet al., 2001).Erectile dysfunction is currently considered as an early clinicalmanifestation of a more generalized vascular disease due to its high prevalencein patients with cardiovascular risk factors including diabetes, hypertension,hyperlipidemia, and tobacco abuse (Sullivan et al., 1999; Montorsi et al., 2003).Erectile dysfunction is a common complication and an important cause ofdecreased quality of life in men with diabetes, and its prevalence is three timeshigher in type 1 and type 2 diabetic patients than in the general population(Hakim and Goldstein, 1996; Vickers and Wright, 2004). About 50% of thesepatients exhibit suboptimal responses to oral phophodiesterase 5 inhibitors(Vickers and Wright, 2004; Vardi, 2009).Growing epidemiological evidence associates the subsequent risk oferectile dysfunction with the presence of risk factors for coronary artery diseasesuch as obesity, hypertension, and dislipidemia (Montorsi et al., 2003; Montorsiet al., 2006). On the other hand, the rate of erectile dysfunction in patients withcoronary artery disease is as high as 42–57%, and the incidence of erectiledysfunction in diabetic patients with silent ischemia is 34.8% versus 4.7% inthose without silent ischemia (Gazzaruso et al., 2004; Montorsi et al., 2003;Montorsi et al., 2006). This has recently led to the suggestion that erectiledysfunction could be a potential marker for silent coronary artery disease in type2 diabetes mellitus patients (Gazzaruso et al., 2004; Montorsi et al., 2006).Elevation of free intracellular Ca2+ allows arterial vasoconstriction, andagonists binding α1-adrenoceptors activate a G protein-coupled pathway tostimulate both Ca2+ mobilization from intracellular stores and Ca2+ entry fromextracellular fluid (Berridge, 2003). However, in many resistance vessels asustained rise in intracellular Ca2+ concentration ([Ca2+]i) is mainly generated viaagonist-induced Ca2+ entry through both dihydropyridine-sensitive L-type andnon-L-type Ca2+ channels (McFadzean and Gibson, 2002; Sanders, 2001). Ca2+entry through non-L-type Ca2+ channels may in turn include voltageindependentstore (SOC)- and receptor (ROC)-operated Ca2+ channels(McFadzean and Gibson, 2002). Elevation of [Ca2+]i in vascular smooth muscle(VSM) cells is considered a classic trigger for force development in smoothmuscle through activation of myosin light chain (MLC) kinase (MLCK) andsubsequent phosphorylation of the 20-kDa regulatory myosin light chain(MLC20), resulting in shortening of VSM. Regulation of the activity of MLCphosphatase (MLCP) mediates the myofilaments sensitivity to Ca2+ (Somlyoand Somlyo, 2000). Phosphorylation of the MYPT1 subunit of MLCP inhibits itsactivity, increasing the Ca2+ sensitivity of the contractile apparatus for a given[Ca2+]. It is well established that MLCP can be inhibited via activation of thesmall G protein RhoA. On activation, RhoA translocates to the membranewhere it activates Rho kinase, which can inhibit MLCP by phosphorylation ofMYPT1 (Feng et al., 1999). Recent studies have shown relevant mechanisms ofCa2+ sensitization involving RhoK in the contraction of corpus cavernosumsmooth muscle and that the NO-cGMP pathway can modulate Ca2+ sensitivityto elicit relaxation (Takahashi et al., 2001; Wang et al., 2002). Whereas severalphysiological issues concerning innervation, receptors, K+ channels, gapjunctionsand local factors which regulate penile smooth muscle tone are wellestablished (Andersson and Wagner, 1995; Prieto et al., 1998; Simonsen et al., 2002), the Ca2+ signaling mechanisms underlying the vasoconstriction oferectile tissue are poorly understood.The purpose of the present study was four-fold: 1) To investigate theCa2+ signaling mechanisms coupled to the α1-adrenoceptor- and to thethromboxane receptor (TP)-mediated vasoconstriction in rat penile smallarteries; 2) To assess the role of Rho kinase in both Ca2+ sensitization and inthe modulation of Ca2+ entry through plasma membrane; 3) To establish ananimal model for a direct assessment of Ca2+ handling in the penile diabeticvasculopathy by determining vascular structure and reactivity of penile arteriescompared to coronary arteries in the obese Zucker rat; 4) To determine whetherimpaired Ca2+ signaling may contribute to the vascular dysfunction ofprediabetic penile arteries.In order to clarify these aims we used first- or second-order branches ofthe rat dorsal penile artery from Wistar rats (12-14 wk), and second-orderbranches of the left descending coronary arteries and dorsal penile arteriesfrom obese Zucker rats (OZR) and their control counterparts [lean Zucker rats(LZR)] (17-18 wk). In these vessels, studies of vascular reactivity with/withoutsimultaneous measurements of intracellular Ca2+ levels were performed.Western blot analysis was used to determinate the total protein expression aswell as immunohistochemistry to study the presence and distribution of proteinson stained arterial cross sections. Determination of lumen, media, and vesselareas was performed by morphometric analysis.The main findings of this study were:1. The α1-adrenergic and TP-mediated vasoconstriction of rat penile arteries iscoupled to Ca2+ entry through voltage-dependent L-type and receptoroperatedCa2+ channels with a minor role of Ca2+ mobilization fromintracellular stores.2. Rho-kinase is involved in the regulation of Ca2+ entry through receptoroperatedCa2+ channels activated by α1-adrenoceptor stimulation, but not byTP activation. 3. Passive depletion of intracellular Ca2+ stores revealed a capacitative Ca2+entry in penile arteries neither coupled to contraction nor Rho-kinasemediated.4. The α1-adrenergic vasoconstriction in penile arteries involves Ca2+sensitization mechanisms mediated by protein kinase C, tyrosine kinase, andRho-kinase.5. TP-mediated Ca2+ sensitization under TP activation in erectile tissue involvesphosphorylation of the MYPT1 subunit of MLCP through Rho-kinase.6. In penile arteries from obese Zucker rats we found a correlation betweenvascular remodeling and endothelial dysfunction, whereas structure waspreserved in coronary arteries despite a developing endothelial dysfunctiondepicted by an impaired NO basal production.7. In both penile arteries and coronary arteries from animals with MetabolicSyndrome vasoconstriction is augmented in part due to impairment of basalNO release and the effect of oxidative stress.8. A mayor role for Rho-kinase-mediated Ca2+ sensitization coupled to a1-adrenergic vasoconstriction at the expense of reduced Ca2+ influx is found inpenile arteries from obese Zucker rats in contrast to LZR.

Final conclusion:In summary, under situations of vascular risk, Ca2+ handling is impaired inpenile arterial tissue with an increase in Ca2+ sensitization mechanisms coupledto a1-adrenergic vasoconstriction at the expense of reduced Ca2+ influx,probably shifted to cell growth and proliferation processes of arterial wall. Acrucial role for Rho-kinase during this change places Rho-kinase inhibitors asan alternative therapeutic target for erectile dysfunction. On the other hand, theseverity of the structural and functional abnormalities in penile arteries during Metabolic Syndrome might anticipate the vascular dysfunction of the coronaryvascular bed and reinforces the current clinical concept that considers erectiledysfunction as a clinical manifestation of both endothelial dysfunction and cardiovascular disease, suggesting that erectile dysfunction could beconsidered as an early sign of other severe vascular pathologies.