Contractile Signaling
The vast majority of time, the penis is maintained in the flaccid state through active contraction of penile arterioles. The release of norepinephrine (NE) from sympathetic nerve terminals activates arteriolar and cavernosal α-adrenergic receptors. Subsequent increases in intracellular Ca concentration ([Ca]i) result in the activation of myosin light-chain kinase (MLCK) and phosphorylation of myosin light chain (MLC), enabling actin–myosin cross-bridge cycling. In addition to the Ca-dependent contractile mechanism, Ca-sensitizing pathways, such as ROCK- and protein kinase C (PKC)-mediated signaling, can promote contraction through the inhibition of MLC phosphatase or the direct stimulation of MLC phosphorylation. Both Ca-dependent and Ca-sensitizing signaling can be activated by NE or other agonists, including endothelin-1 (ET-1), serotonin (5-HT) and angiotensin-II (Ang-II).
Upstream Signaling: NE, ET-1, 5-HT and Ang-II
NE It is generally accepted that penile detumescence and flaccidity are achieved mainly by constant sympathetic input. Both cavernous smooth muscle (CSM) and the smooth muscle of the penile arteries and veins are rich in sympathetic innervation. Upon activation, the sympathetic nerve terminals release NE, which binds to and stimulates α-adrenoceptors on the smooth muscle membrane. The activation of α-adrenoceptors triggers a series of intracellular signaling pathways involving both Ca-dependent and Ca-sensitizing mechanisms, to induce contraction.
ET-1 ET-1, a member of the ET family of peptides, is among the strongest vasoconstrictors known. ET-1 is produced primarily in the endothelium and plays an important role in vascular homeostasis. In the corpus cavernosum (CC), ET-1 elicits slow-developing but strong, long-lasting contractions. Smooth muscle cells including CSM cells express two subtypes of ET-1 receptors: ETA and ETB. The binding of ET-1 to ETA increases vasocontraction, whereas the binding of ET-1 to ETB leads to vasorelaxation via the release of NO. In the CC, ETA expression is dominant over ETB, and therefore, ET-1 mainly induces CSM contraction. Similar to NE, ET-1-induced CSM contraction is mediated by an increase in both [Ca]i and Ca sensitization. Evidence indicates that changes in the ET-1 pathway are involved in the pathophysiology of ED. Chang et al. found that the expression of ETA receptors was significantly upregulated in the CSM of diabetic rabbits. Sullivan et al. reported a significant decrease in ETB receptor binding sites in cavernosal tissue from hypercholesterolemic rabbits, and a significant increase in ETB receptor binding sites in cavernous tissue of diabetic rabbits.
5-HT 5-HT is a monoamine neurotransmitter that has a variety of functions in the central nervous system as well as peripheral tissues. In the CC, 5-HT released from the serotonergic nerve fibers has a physiological role in the maintenance of penile flaccidity and the initiation of detumescence. Several studies showed that 5-HT induced contraction of isolated penile tissue, which could be blocked by 5-HT receptor antagonists. Further, pre-incubation with ROCK inhibitor Y-27632 attenuated maximum contraction induced by 5-HT of penile tissue, indicating that RhoA/ROCK pathway is a mediator of 5-HT-induced contraction. Evidence from human pulmonary arteries showed that Ca influx and release from the intracellular Ca store may also be involved in 5-HT-induced contraction.
Ang-II Being an important component of the renin–Ang system, Ang-II is converted from Ang-I by Ang-converting enzyme (ACE) predominately in the lung. Ang-II binds to Ang receptors on the membranes of smooth muscle cells and other cell types, causing contraction via both Ca-dependent and Ca-sensitizing mechanisms, similar to the other agonists. Becker et al. reported that Ang-II levels were 30% higher in the cavernous blood than that in the systemic blood, indicating that Ang-II is produced locally in the CC. This is further supported by the findings that ACE is expressed in the endothelial cells of canine CC. The dynamic changes in Ang-II levels in the cavernous blood during different penile states suggest that Ang II plays a physiological role in regulating penile tone. In organ-bath studies, Ang-II evoked dose-dependent contraction of human and rabbit CC strips. In vivo studies demonstrated that intracavernosal injection (ICI) of Ang-II terminated spontaneous erections, whereas an Ang II receptor blocker, losartan, increased the intracavernosal pressure, in a dose-dependent manner, in anesthetized dogs.
Intracellular Signaling
Ca-dependent PathwaysRegulation of [Ca]i: It has been widely accepted that elevated [Ca]i is critical for maintaining smooth muscle in a contracted state. The increase in [Ca]i could be a result of: (1) increased Ca release from the intracellular store-sarcoplasmic reticulum (SR); (2) increased Ca influx, mainly through the L-type voltage-gated Ca channels (VGCCs); and/or (3) inhibited Ca removal.
Carelease from the SR—There are two types of Ca channels in the SR membrane: inositol trisphosphate (IP3) and ryanodine receptors. The binding of agonists (NE and others) to their receptors on the cell membranes activates phospholipase C, which leads to the production of IP3 and diacylglycerol. IP3 then binds to IP3 receptors on the SR membrane and triggers the release of Ca. These Ca transients activate Ca-dependent Cl channels and depolarize the membrane, and in turn, open the VGCCs. The opening of ryanodine receptors is Ca-dependent, through the process of Ca-induced Ca release, resulting in a further increase in [Ca]i. The role of Ca release through ryanodine receptors seems to be more complicated: whereas evidence shows that they function similarly as the IP3-mediated Ca release (that is, mediating contraction), other studies indicate that they may activate Ca-dependent K channels, which in turn causes hyperpolarization of smooth muscle cells leading to relaxation.
Cainflux through the L-type VGCCs—Although other types of Ca channels are involved in Ca influx in CSM cells, the L-type VGCCs are believed to play the leading role in mediating CSM contraction. This is supported by the finding that L-type VGCC blockers relax cavernous tissue strips contracted with NE. The L-type VGCCs open when the membrane potential increases from the resting level (−90 mV) to −30 mV, resulting from Cl influx induced by Ca release from the SR, or Ca influx via other types of Ca channels, including T-type VGCC. Furthermore, L-type VGCCs can also be activated when they are phosphorylated by PKC (discussed below). The closure of the L-type VGCC is induced by K efflux via Ca-dependent K channels, and the NO/cGMP pathway.
Caremoval—Removal of the [Ca]i after it has risen, and maintaining a low background [Ca]i, is mainly achieved by pumping Ca to the SR lumen by the sarco(endo)plasmic reticulum Ca-ATPases, or to the extracellular spaces by the Na–Ca exchangers and the plasma membrane Ca-ATPases. It is clear that [Ca]i removal can be promoted by NO/cGMP pathway via activating sarco(endo)plasmic reticulum Ca-ATPases, Na–Ca exchangers and plasma membrane Ca-ATPases. The activity of the sarco(endo)plasmic reticulum Ca-ATPases in the CC is significantly higher than that in the bladder and urethra, and can be significantly downregulated by castration. The role of the Na–Ca exchangers and the plasma membrane Ca-ATPases in the CSM is unclear.
MLCK and MLC: [Ca]i binds to calmodulin, and in turn, the Ca/calmodulin complex activates MLCK. MLCK then phosphorylates the regulatory unit of MLC, allowing it to activate myosin ATPase. Activity of myosin ATPase permits ratcheting of myosin and actin myofilaments and muscular contraction. On the contrary, phosphorylated MLC can be dephosphorylated by MLC phosphatase, resulting in a reversal of contraction. At basal levels of tone, only ~10% of the MLC in the CC exists in a phosphorylated state, a significantly lower level than that in the bladder (25%). Upon maximal stimulation by phenylephrine, the MLC in the CC reaches a phosphorylation level of 23%. It has been shown that agonists (NE, ET-1, and so on) also augment G-protein-dependent downregulation of MLC phosphatase activity, resulting in an increase in the level of MLC phosphorylation, which can be reversed by activating the NO-cGMP pathway. The Ca-dependent pathways regulating smooth muscle contraction are summarized in Figure 1.
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Figure 1.
Ca-dependent pathways in smooth muscle cells. The binding of agonists (NE, ET, and so on) to their receptors on the cell membrane induces Ca release from the intracellular stores and Ca influx through VGCC. Subsequent increases in [Ca]i result in the activation of myosin light-chain kinase and phosphorylation of myosin light chain, promoting smooth muscle contraction. Abbreviations: ET, endothelin; CDCC, Ca-dependent Cl channels; CaM, calmodulin; DAG, diacylglycerol; IP3, inositol trisphosphate; IP3R, IP3 receptors; MLC, myosin light chain; MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; NE, norepinephrine; P, phosphate; PKC, protein kinase C; PLC, phospholipase C; R, receptors; SR, sarcoplasmic reticulum; VGCC, voltage-gated calcium channels.
Ca-sensitizing PathwaysRhoA/Rho-kinase:RhoA is a low-molecular-weight G protein, which is active in the GTP-bound state. The activation of RhoA requires both its translocation to cellular membrane and the post-translational addition of a geranylgeranyl phosphate onto it. RhoA has numerous downstream targets, a predominant one of which is the ser/thr kinase, ROCK. ROCK has been shown to induce calcium sensitization of smooth muscle by phosphorylating the myosin binding subunit of MLC phosphatase, leading to the inhibition of MLC phosphatase activity. Some studies also suggest that ROCK may phosphorylate MLC directly. ROCK-mediated inhibition of MLC phosphatase leads to the maintenance of the phosphorylated state of MLC, promoting vascular smooth muscle contraction. Numerous studies have demonstrated that the inhibition of RhoA/ROCK-mediated Ca sensitization induces the relaxation of smooth muscle. In a recent study, Li et al. found that the penile RhoA/ROCK pathway was upregulated in diabetic rats, and chronic treatment with the ROCK inhibitor fasudil could restore erectile function by normalizing the Akt-driven pathway, indicating that the RhoA/ROCK pathway plays a pivotal role in the pathogenesis of diabetic ED.
There are two isoforms of ROCK: ROCK1 (ROKβ) and ROCK2 (ROKα). In humans, ROCK1 and ROCK2 genes are located separately on chromosomes 18 and 2, respectively. ROCK1 is preferentially expressed in inflammatory cells, whereas ROCK2 is highly expressed in vascular smooth muscle cells. Wang et al. reported that whereas a balance of ROCK1 and ROCK2 activities is required to regulate vascular smooth muscle actin cytoskeletal structure, ROCK2 is the predominant isoform that regulates vascular smooth muscle cell contractility. Both ROCK1 and ROCK2 are expressed in human and animal CC. Elevated expression of the ROCK2 protein was found in the cavernous tissue of spontaneous hypertensive rats and rats that had undergone cavernous nerve injury, in line with the findings that ROCK2 plays the major role in regulating vascular smooth muscle cell contractility. However, several studies indicate that the expression of ROCK1, rather than ROCK2, was significantly increased in penile tissues from different diabetic animal models. In rabbits with bladder outlet obstruction, Chang et al. found that the expression of both isoforms of ROCK in the CC is increased. The etiology-dependent changes in the expression of ROCK1 and ROCK2 indicate that they might play different roles in the pathophysiology of ED, which may have implications in the development of therapeutic options.
Telokin: Telokin, also known as kinase-related protein, is a 17-kDa smooth muscle-specific protein whose sequence is identical to the C-terminal domain of MLCK. It has been shown that telokin decreases smooth muscle contractility by inhibiting the phosphorylation of the regulatory unit of MLC by the MLCK. Increasing evidence shows that telokin also activates MLC phosphatase, and therefore, leads to Ca desensitization. Telokin knockout mice exhibit decreased MLC phosphatase activity, resulting in increased Ca sensitivity in intestinal smooth muscle. The activity and function of telokin in the cavernous tissue remains unclear.
PKC: PKC is a family of enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino-acid residues on these proteins. Activated by signals such as diacylglycerol or Ca, PKC regulates smooth muscle tone via complex and diverse signal-transduction cascades. First, PKC regulates the activity of L-type VGCCs, non-selective cation transient receptor potential channels, Ca-activated K channels and ATP-sensitive K channels by phosphorylating them. The function of PKC could be either activating or inhibiting these ion channels, depending on the cell type, the PKC isoforms involved and the concentration of PKC agonist. Another mechanism by which PKC regulates smooth muscle tone is to increase Ca sensitization via phosphorylation of CPI-17 (discussed next). In addition, PKC was found to inhibit NO synthase activity. Increased PKC activity in diabetic human cavernous tissue has been reported, indicating that the alteration of PKC activity might be involved in the pathogenesis of ED. However, Jin et al. failed to detect any significant effect of a PKC activator or PKC inhibitors on the tone of mouse cavernous tissue, although there were significant effects on mouse aorta.
CPI-17: CPI-17, known as PKC potentiated inhibitory protein of protein phosphatase-1, is a 17-kDa protein that can be phosphorylated primarily by PKC, although other kinases, such as ROCK and p21-activated protein kinase, have been suggested to phosphorylate CPI-17 as well. Phosphorylation at Thr-38 greatly increases the inhibitory potency of CPI-17, which in turn inhibits MLC phosphatase activity, leading to increased phosphorylation of MLC and contraction of smooth muscle. The phosphorylation of CPI-17 was diminished in decompensated bladder tissue, indicating that the activity level of CPI-17 is correlated with smooth muscle contractility. CPI-17 expression was detected in human and rabbit CC; however, its role in penile erection remains to be determined. Figure 2 illustrates the Ca-sensitizing pathways regulating smooth muscle contraction.
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Figure 2.
Ca-sensitizing pathways in smooth muscle cells. The binding of agonists (NE, ET, and so on) to their receptors on the cell membrane induces activation of RhoA. RhoA-GTP in turn activates ROCK, leading to the inhibition of MLC phosphatase activity by phosphorylation of the myosin binding subunit of MLC phosphatase. Furthermore, both PKC and ROCK can phosphorylate and activate CPI-17, which also inhibits MLC phosphatase activity, to promote smooth muscle contraction. Abbreviations: ET, endothelin; DAG, diacylglycerol; IP3, inositol trisphosphate; MLC, myosin light chain; MLCP, myosin light-chain phosphatase; MLCK, myosin light-chain kinase; NE, norepinephrine; P, phosphate; PLC, phospholipase C; PKC, protein kinase C; R, receptors; ROCK, Rho-kinase.