Search

Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC17) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC17aand SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC17b, and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.

Diabetes results in a myriad of vascular complications, often referred to as diabetic vasculopathy, which encompasses both microvascular [erectile dysfunction (ED), retinopathy, neuropathy, and nephropathy] and macrovascular complications (hypertension, coronary heart disease, and myocardial infarction). In diabetic animals and patients with ED, there is decreased opiorphin or opiorphin-related gene expression in corporal tissue. Both opiorphin and the rat homologous peptide sialorphin are found circulating in the plasma. In the present study, we investigated if diabetes induced changes in plasma sialorphin levels and if changes in these levels could modulate the biochemistry and physiology of vascular smooth muscle. We show that circulating sialorphin levels are reduced in a rat model of type I diabetes. Intracorporal injection of plasmids expressing sialorphin into diabetic rats restores sialorphin levels to those seen in the blood of nondiabetic animals and results in both improved erectile function and blood pressure. Sialorphin modulated the ability of C-type natriuretic peptide to relax both corporal and aortic smooth muscle strips and of bradykinin to regulate intracellular calcium levels in both corporal and aortic smooth muscle cells. We have previously shown that expression of genes encoding opiorphins is increased when erectile function is improved. Our findings thus suggest that by affecting circulating levels of opiorphin-related peptides, proper erectile function is not only an indicator but also a modulator of overall vascular health of a man.

Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism.

The Ca2+-sensitive K+channel (maxi-K+) is an important modulator of corporal smooth muscle tone. The goal of these studies was twofold: 1) to determine the feasibility of transfecting corporal smooth muscle cells in vivo with the hSlo cDNA, which encodes for the human smooth muscle maxi-K+channel, and 2) to determine whether transfection of the maxi-K+channel would affect the physiological response to cavernous nerve stimulation in a rat model in vivo. Intracorporal microinjection of pCMVβ/Lac Z DNA in 10-wk-old rats resulted in significant incorporation and expression of β-galactosidase activity in 10 of 12 injected animals for up to 75 days postinjection. Moreover, electrical stimulation of the cavernous nerve revealed that, relative to the responses obtained in age-matched control animals ( N = 12), intracavernous injection of naked pcDNA/ hSlo DNA was associated with a statistically significant elevation in the mean amplitude of the intracavernous pressure response at all levels of current stimulation (range 0.5–10 mA) at both 1 mo ( N= 5) and 2 mo ( N = 8) postinjection. Furthermore, qualitatively similar observations were made at 3 mo ( N = 2) and 4 mo ( N = 2) postinjection. These data indicate that naked hSlo DNA is quite easily incorporated into corporal smooth muscle and, furthermore, that expression is sustained for at least 2 mo in corporal smooth muscle cells in vivo. Finally, after expression, hSlo is capable of measurably altering nerve-stimulated penile erection. Taken together, these data provide compelling evidence for the potential utility of gene therapy in the treatment of erectile dysfunction.

Ratio imaging using the calcium-sensitive probe fura-2 was employed to study intracellular calcium concentrations and intercellular calcium flux through gap junctions in homogeneous vascular smooth muscle cell cultures derived from the human corpora cavernosa. Microinjection techniques demonstrated that fura-2 free acid was freely diffusible through gap junctions between cultured cells. The resting intracellular calcium level in fura-2-loaded cells was 176.9 +/- 10.5. A robust increase in intracellular calcium was seen in response to both phenylephrine and the calcium ionophore A23187. Microinjection of Ca2+ into individual smooth muscle cells always resulted in significant, although temporally delayed, increases in intracellular calcium levels in adjacent cells; this intercellular calcium flux was reversibly blocked by inhibition of gap junctional communication with 2 mM heptanol. However, although microinjection of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] into individual smooth muscle cells always produced significant increases in intracellular calcium levels in the injected cell, the intercellular spread of Ca2+ in response to Ins(1,4,5)P3 was more variable than for Ca2+ injections. These studies demonstrate that Ca2+, and perhaps Ins(1,4,5)P3 as well, can diffuse between smooth muscle cells through gap junction channels.

Kinetic studies were conducted on the contractile response elicited by phenylephrine (PE) activation of the alpha 1-adrenergic receptor subtype in vascular smooth muscle isolated from the corpus cavernosum of impotent men. PE-induced contractions were separated into distinct phasic and tonic components, and the tonic portion was analyzed using a first-order rate equation to determine the maximal rate constant for onset of contraction (kobs max) and the maximum amplitude of the steady-state contractile response (Req max). The kobs max value in tissues from insulin-dependent diabetic patients was significantly greater than that in tissues from either noninsulin-dependent diabetics or nondiabetics. Additionally, the mean kobs max value in older patients (60-70 yr) was significantly greater than the mean kobs max value in younger patients (32-59 yr). Significant diabetes-related, but not age-related, alterations were also found in Req max. The observed changes in contractility resulted in dramatic age- and pathology-dependent alterations in the initial rate and/or magnitude of PE-induced response generation. These kinetic studies extend our previous observations at steady state and provide further evidence for heightened corporal tissue tone in the etiology of erectile dysfunction.