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Effects of C-peptide on expression of eNOS and iNOS in human cavernosal smooth muscle cells
BASIC SCIENCE
EFFECTS OF C-PEPTIDE ON EXPRESSION OF eNOS AND iNOS IN HUMAN CAVERNOSAL SMOOTH MUSCLE CELLS HAIKUN LI, LIPING XU, JOSEPH C. DUNBAR, CHIRPRIYA B. DHABUWALA, ANDERS A. F. SIMA
AND
ABSTRACT Objectives. To investigate the role of C-peptide alone or in conjunction with insulin on the expression of nitric oxide synthase (NOS) in human corpus cavernosum smooth muscle cells (HCSMCs). Erectile dysfunction, among diabetic patients, is a significant health problem. The specific causes of erectile dysfunction are unknown. It has been suggested that impairment of penile relaxation is related to a reduction of penile NOS. Plasma levels of C-peptide and insulin are decreased in individuals with type 1 diabetes and late-stage type 2 diabetes. Methods. Primary cultures were initiated from explants of HCSMCs. Confluent cells at passages 2 to 4 were assigned to one of four groups with the following incubation conditions: (a) 27 mM glucose, (b) 27 mM glucose and insulin, (c) 27 mM glucose and human recombinant (hr)C-peptide, and (d) 27 mM glucose, insulin, and hrC-peptide. After 24 hours, total RNA and protein were extracted from cells and subjected to reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. Intracellular Ca2⫹ was examined under the four conditions, using the Fura 2 method. Results. The least expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in HCSMCs was observed in cells exposed to 27 mM glucose alone. Increased expression of eNOS and iNOS was found after treatment with insulin or hrC-peptide alone, and the maximal expression of eNOS and iNOS was detected in HCSMCs exposed to both insulin and hrC-peptide. Western blot analyses using eNOS and iNOS antibodies confirmed the RNA data. These effects are likely mediated by the insulin-induced and/or C-peptide-induced increase in intracellular Ca2⫹. Conclusions. Our results demonstrated that C-peptide, in the presence of insulin, increases the expression of iNOS and eNOS in HCSMCs. These results suggest that C-peptide, especially in conjunction with insulin, may have beneficial effects on cavernosal smooth muscle relaxation. UROLOGY 64: 622–627, 2004. © 2004 Elsevier Inc.
E
rectile dysfunction (ED) is a significant health problem in the United States and develops in 35% to 75% of men with diabetes mellitus.1–3 Although ED is not life threatening, it has strong negative psychological effects on a man already burdened by diabetes. The specific causes of ED are unknown. The functional regulation of penile smooth muscle reThis study was supported in part by grants from the Juvenile Diabetes Research Foundation and the Thomas Foundation, Bloomfield Hills, Michigan to A. A. F. Sima. From the Departments of Urology, Physiology, Neurology, and Pathology, Wayne State University School of Medicine, Detroit, Michigan Reprint requests: Anders A. F. Sima, M.D., Ph.D., Department of Pathology, Wayne State University School of Medicine, Gordon H. Scott Hall, 540 East Canfield Avenue, Detroit, MI 48201 Submitted: June 10, 2003, accepted (with revisions): May 5, 2004
622
© 2004 ELSEVIER INC. ALL RIGHTS RESERVED
laxation is of prime importance for the development of normal erections. Among the neurotransmitters involved in cavernosal smooth muscle relaxation, nitric oxide (NO), synthesized by nitric oxide synthase (NOS), plays an essential role in ensuring normal penile erection.4 – 6 The potential sources of NO in the penis include neurons, sinusoidal endothelium, and corporeal smooth muscle cells.6 In diabetes, it has been suggested that impaired relaxation of blood vessels and the trabecular meshwork of smooth muscle corpora cavernosum is related to a reduction in penile NOS activity/expression, with resultant decreases in NO production.6 – 8 The discovery of insulin has greatly improved the metabolic control of diabetes; however, microvascular complications are still common among diabetic patients despite intensive insulin treatment 0090-4295/04/$30.00 doi:10.1016/j.urology.2004.05.005
and well-controlled glucose levels.9,10 C-peptide is a 31-amino acid peptide cleaved from pro-insulin and co-secreted with insulin.11 Plasma levels of Cpeptide and insulin are markedly decreased in individuals with type 1 diabetes and in those with the late stage of type 2 diabetes.12 Recent studies have demonstrated that C-peptide possesses biologic activities of its own.13–17 However, the role of C-peptide in the expression of penile NOS has not been studied. The present study was designed to test the hypotheses that C-peptide alone, or combined with insulin, influences the expression of NOS and that this is associated with intracellular Ca2⫹ regulation. First, we investigated the role of C-peptide in the expression of inducible NOS (iNOS) and endothelial NOS (eNOS) in human cavernosal smooth muscle cells (HCSMCs) in vitro. Second, we examined its regulatory role on intracellular Ca2⫹. MATERIAL AND METHODS CELL CULTURE AND TREATMENT HCSMCs were cultured as described by Rajasekaran et al.18 In brief, primary cultures were initiated using explants of human corpora cavernosa (2 mm3), obtained from nondiabetic patients receiving penile implants. The tissue was placed in Dulbecco’s growth media containing 20% fetal bovine serum for 5 days. The cells were then incubated with fresh Dulbecco’s growth media until confluence. The presence of smooth muscle cells was confirmed by smooth muscle actin staining. Monolayers of cells (80% to 90% confluence) at passages 2 to 4 were assigned to one of four groups incubated with (a) 27 mM glucose, (b) 27 mM glucose and insulin (500 U/mL, Sigma Chemical, St. Louis, Mo), (c) 27 mM glucose and human recombinant (hr)C-peptide (10 ng/mL, Schwarz Pharma GMBH, Monheim, Germany), or (d) 27 mM glucose, insulin, and hrC-peptide. After 24 hours of incubation, protein and total RNA was extracted and the expression of iNOS and eNOS detected by Western blotting and reverse transcriptase-polymerase chain reaction, respectively.
DETERMINATION OF NOS BY WESTERN BLOTTING Two NOS isoforms, iNOS and eNOS, were studied. Cells were homogenized in a lysis buffer (10 mM Tris-HCl, 1 mM ethylene-diamine-tetra-acetic acid or ethylenediaminetetraacetic acid, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktails). The homogenates were then centrifuged at 15,000g for 20 minutes at 4°C. Supernatants were collected, and the protein concentration was determined using a Bio-Rad protein assay kit. A total of 80 g protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. Equal loading of protein was ascertained by the Ponceau S stain. The membrane was then incubated with anti-iNOS or anti-eNOS antibody (Transduction Laboratories, San Diego, Calif) at a dilution of 1:2000 at 4°C overnight, and with a secondary antibody at a dilution of 1:2000 for 90 minutes. Immune complexes were detected with the enhanced chemiluminescence system (Amersham, Arlington Heights, Ill). The results were quantified with a scanning densitometer using an image analysis system with National Institutes of Health Image, version 1.55, software (Image Research, St. Catharines, Canada). UROLOGY 64 (3), 2004
DETERMINATION OF NOS BY REVERSE TRANSCRIPTASEPOLYMERASE CHAIN REACTION Total RNA was isolated from HCSMCs after 24 hours of treatment using an RNA isolation kit (Gibco, Gaithersburg, Md). cDNA was synthesized from 3 g total RNA with reverse transcriptase (SuperScript II, Gibco-BRL). Reverse transcription was performed at 42°C for 1 hour after incubation at 95°C for 5 minutes. cDNA amplification was performed according to the following temperature profile: 94°C for 1 minute; 60°C for 1 minute; and 72°C for 1.5 minutes. At the end of 35 cycles, the reaction was prolonged for 10 minutes at 72°C, and then 5 L was analyzed on a 1.5% agarose gel. Beta-actin was used as the control. Comparisons between the different conditions were made within the linear range of amplification of eNOS, iNOS, and beta-actin. The sequences of primers were 5⬘-GTGATGGCGAAGCGAGTGAAG-3⬘ and 5⬘-CCGAGCCCGAACACACAGAAC-3⬘ for eNOS; and 5⬘-TCCGAGGCAAACAGCACATCA-3⬘ and 5⬘-GGGTTGGGGGTGTGGTGATGT-3⬘ for iNOS; and 5⬘-AACCGCGAGAAGATGACCCAGATCATGTTT-3⬘ and 5⬘-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3⬘ for beta-actin.
MEASUREMENT OF INTRACELLULAR Ca2ⴙ Intracellular Ca2⫹ concentration was measured in cells exposed to normal (5 mM) or high (27 mM) glucose level with or without insulin (500 U/mL) or insulin plus hrC-peptide (10 ng/mL), using Fura 2 (TEF Labs, Austin, Tex) and dual excitation wavelength fluorescence microscopy.19,20 In brief, cells were incubated with physiologic saline solution containing 7 M Fura 2 acetoxymethyl ester, 0.09 g/dL dimethyl sulfoxide and 0.018 g/dL Pluronic F-127 (Molecular Probes, Eugene, Ore) for 60 minutes at 23°C. Cells were illuminated alternatively with excitation wavelengths of 340 and 380 nm. The emission wavelength was 510 nm. Background corrected data were collected using the Felix software package (Photon Technology International, Monmonth Junction, NJ).
STATISTICAL ANALYSIS
The data are presented as the mean ⫾ SEM. Statistical analysis was performed using analysis of variance followed by Newman-Keuls post hoc test. Probability values of less than 0.05 were considered statistically significant.
RESULTS EXPRESSION OF NOS mRNA Polymerase chain reaction analysis detected fragments of approximately 460 and 420 base pairs specific for iNOS and eNOS, respectively. The least expression of iNOS and eNOS mRNA was observed in HCSMCs treated with 27 mM glucose alone. A small increase in the levels of iNOS and eNOS mRNA occurred in cells treated either with insulin or hrC-peptide alone compared with that of 27 mM glucose alone. However, the maximal expression of eNOS and iNOS was detected in HCSMCs in the presence of both hrC-peptide and insulin (Fig. 1). EXPRESSION OF NOS PROTEIN Western blotting showed single bands of 130 kDa and 140 kDa for iNOS and eNOS, respectively. In cells treated with either hrC-peptide or insulin alone, a small increase was observed in the expression of these two isoforms compared with 27 mM 623
FIGURE 1. Expression of iNOS and eNOS mRNA in HCSMCs in primary culture. Lanes 1 to 4 correspond to cDNA fragments amplified from cells treated with 27 mM glucose, 27 mM glucose plus insulin, 27 mM glucose plus hrC-peptide, and 27 mM glucose plus insulin and hrC-peptide, respectively. Data shown from representative experiment of three independent experiments. FIGURE 3. Expression of eNOS protein in HCSMCs in primary culture. (A) Western blot analysis of eNOS expression in HCSMCs. Lanes 1 to 4 correspond to eNOS expression from cells treated with 27 mM glucose (basal), 27 mM glucose plus insulin, 27 mM glucose plus hrC-peptide, or 27 mM glucose plus insulin (Ins) and hrC-peptide, respectively. Data shown from representative experiment of three independent experiments. (B) Quantitative results using densitometric scanner. Data expressed as percentage of basal levels (27 mM glucose) for three independent experiments. *P ⬍ 0.01, **P ⬍ 0.001 versus basal condition; †P ⬍ 0.05 versus insulin or hrC-peptide alone.
FIGURE 2. Expression of iNOS protein in HCSMCs in primary culture. (A) Western blot analysis of iNOS expression in HCSMCs. Lanes 1 to 4 correspond to iNOS expression from cells treated with 27 mM glucose (basal), 27 mM glucose plus insulin, 27 mM glucose plus hrC-peptide, or 27 mM glucose plus insulin (Ins) and hrC-peptide, respectively. Data shown from representative experiment of three independent experiments. (B) Quantitative results using densitometric scanner. Data expressed as percentage of basal levels (27 mM glucose) for three independent experiments. *P ⬍ 0.01; **P ⬍ 0.001 versus basal condition; †P ⬍ 0.005 versus insulin or hrC-peptide alone.
glucose alone. The maximal expression of the two isoforms was detected in cells treated with both hrC-peptide and insulin (Fig. 2A and 3A). Quantitative densitometry from three independent experiments revealed that the level of iNOS in HCSMCs increased by 1.4-fold (P ⬍ 0.05), 1.2-fold (P ⬍ 0.05) and 2.3-fold (P ⬍ 0.001) above basal levels in cells treated with insulin alone, hrC-peptide alone, and both hrC-peptide and insulin, respectively (Fig. 2B). The expression of eNOS was increased by 1.3-fold (P ⬍ 0.05), 1.2-fold (P ⬍ 0.05), and 1.6-fold (P ⬍ 0.001) above the basal levels in cells treated with insulin alone, hrC-pep624
tide alone, and both hrC-peptide and insulin, respectively (Fig. 3B). INTRACELLULAR Ca2ⴙ No differences in intracellular Ca2⫹ were detected between cells incubated with 5 and 27 mM glucose. The addition of insulin alone (500 U/ mL) increased intracellular Ca2⫹ in cells exposed to both glucose concentrations (P ⬍ 0.01 for both). When incubated with both insulin and hrC-peptide (10 ng/mL), a small additional increase (P ⬍ 0.05) in intracellular Ca2⫹ was seen in cells exposed to 5 mM glucose; however, the intracellular Ca2⫹ expression was maximal (P ⬍ 0.001 versus control) in cells exposed to 27 mM glucose and insulin plus hrC-peptide (Fig. 4). COMMENT The present data have demonstrated that C-peptide increases the expression of iNOS and eNOS in HCSMCs. This was further increased in the presence of both C-peptide and insulin, suggesting a synergistic effect on the regulation of NO in HCSMCs. The vasodilatory effect of insulin21,22 has been linked to the release of NO from endothelial cells and to decreased NO production in insulinUROLOGY 64 (3), 2004
FIGURE 4. Intracellular Ca2⫹ concentrations in HCSMCs incubated in either 5 or 27 mM glucose and with 500 U/mL insulin or insulin and 10 ng/mL hrC-peptide. *P ⬍ 0.01; **P ⬍ 0.001 versus respective concentration of glucose alone; †P ⬍ 0.005 versus 27 mM glucose plus insulin and hrC-peptide.
deficient diabetes,23,24 in agreement with the present data. The additional enhancing effect of C-peptide on NO synthesis in the presence of insulin is in keeping with recent clinical and experimental data.25–28 The present data suggest that the potentiating effect of C-peptide on NO synthesis is mediated by an increase in Ca2⫹ influx, also consistent with recent findings.29 The functional regulation of penile smooth muscle relaxation is of prime importance for the development of normal erections. Of the various mediators involved in cavernosal smooth muscle relaxation, NO plays an essential role in ensuring normal penile erections.4 – 6 NO is synthesized by NOS in a reaction that converts arginine and oxygen into citrulline and NO.5 Measurement of NO production has been accomplished by examining the enzyme responsible for NO synthesis (NOS). To date, several isoforms of NOS have been purified and cloned.30 The inducible isoform of NOS (iNOS) is associated primarily with macrophage function and is activated by specific cytokines. The endothelial (eNOS) and neuronal isoforms of NOS are constitutive and are activated, in part, by increased concentrations of intracellular calcium and calmodulin binding. In this study we have demonstrated that the expression of iNOS and eNOS is increased in HCSMCs after 24 hours of treatment with C-peptide, especially in the presence of insulin. These findings suggest that C-peptide may have a beneficial effect on impaired erectile responses. However, the evidence for a direct link between C-peptide deficiency and ED needs to be determined in clinical trials. UROLOGY 64 (3), 2004
C-peptide is a 31-amino acid peptide cleaved from pro-insulin and co-secreted with insulin.11,28 Plasma levels of C-peptide and insulin are markedly decreased in individuals with type 1 diabetes, as well as in those with late-stage type 2 diabetes.12,28 C-peptide was initially believed to have no biologic effects apart from its role in insulin biosynthesis. However, recent findings of the physiologic effects of C-peptide and beneficial effects of C-peptide replacement in patients with type 1 diabetes have prompted renewed interest. Studies have indicated that in patients with type 1 diabetes who lack endogenous C-peptide, the administration of C-peptide improves renal function, stimulates muscle microcirculation and glucose use, improves blood-retinal barrier function, and improves sensory nerve conduction velocity.12–14,31 The mechanism underlying the observed effects of C-peptide is not clear. Previous studies have suggested that C-peptide, by an increase in intracellular Ca2⫹, stimulates eNOS activity,28,29,32 which is in agreement with the present findings showing increased intracellular Ca2⫹ in cells exposed to both insulin and C-peptide. The subcellular mechanism for C-peptide’s ability to stimulate iNOS expression remains unclear. MacNaul and Hutchinson33 have suggested that iNOS may have negative feedback effects on the eNOS. Our findings did not support this notion, because both iNOS and eNOS expression was increased by Cpeptide and increased further when exposed to both hormones, suggesting that C-peptide acts synergistically with insulin. Evidence for specific binding of C-peptide to a cell surface receptor has been demonstrated. It has been suggested by Rigler et al.34 that this is a G protein-coupled receptor. More recent studies have not been able to confirm this, but have suggested that C-peptide may bind to the insulin receptor itself at a different ligand site.35,36 The latter is consistent with C-peptide signaling by way of the insulin signaling pathways and its enhancing effect on insulin signaling intermediaries in the presence of insulin.35–37 eNOS has been identified in a variety of cell types in addition to the endothelial cell.38 Hung et al.39 reported that rat cavernosal smooth muscle cells express a Ca2⫹-calmodulin dependent constitutive form of NOS (eNOS) in culture. The results from Rajasekaran et al.18 have also shown that HCSMCs in culture express both eNOS and iNOS. In our study, we have demonstrated that HCSMCs, when treated with C-peptide alone or in conjunction with insulin, express both eNOS and iNOS in culture. These observations suggest that cavernosal smooth muscle cells have the ability to synthesize their own NO. Although unlikely, it cannot be totally excluded that this could potentially be induced by in vitro conditions. Thus, C-peptide may 625
exert its function through NO-mediated mechanisms in these cells. Additional support for the idea that C-peptide’s effects may be mediated by a NOmediated pathway is provided by the observation that C-peptide induces a concentration-dependent dilation of rat skeletal muscle arterioles mediated by NO.27 Moreover, C-peptide also stimulate eNOS, with release of NO in a concentration-dependent manner from aortic endothelial cells, effects that are abolished by NOS inhibitors.32 CONCLUSIONS We have demonstrated that C-peptide, in the presence of insulin, increases the expression of iNOS and eNOS in HCSMCs, probably by way of a Ca2⫹-mediated mechanism. This suggests that Cpeptide, especially in conjunction with insulin, may play a regulatory role through NO-mediated mechanisms on HCSMCs, and that its replacement in patients with insulin-dependent diabetes may have therapeutic effects. ACKNOWLEDGMENT. To Dr. Suresh Sikka for providing the human cavernosal smooth muscle cells. REFERENCES 1. Krane RJ, Goldstein I, and Saenz De Tejada I: Impotence. N Engl J Med 321: 1648 –1659, 1989. 2. Kolodny RC, Kahn CB, Goldstein HH, et al: Sexual dysfunction in diabetic men. Diabetes 23: 306 –309, 1974. 3. Feldman HA, Goldstein I, Hatzichristou DG, et al: Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol 151: 54 –61, 1994. 4. Trigo-Rocha F, Hsu GL, Donatucci CF, et al: The role of cyclic adenosine monophosphate, cyclic guanosine monophosphate, endothelium and nonadrenergic, noncholinergic, neurotransmission in canine penile erection. J Urol 149: 872– 877, 1993. 5. Lowenstein CJ, Dinerman JL, and Snyder SH: Nitric oxide: a physiologic messenger. Ann Intern Med 120: 227– 237, 1994. 6. Burnett AL: Nitric oxide in the penis: physiology and pathology. J Urol 157: 320 –324, 1997. 7. Saenz de Tejada I, Goldstein I, Azadzoi K, et al: Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men impotence. N Engl J Med 320: 1025–1030, 1989. 8. Seftel AD, Vaziri ND, Ni Z, et al: Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through iNOS or eNOS. Urology 50: 1016 –1026, 1997. 9. Tooke JE: Microvasculature in diabetes. Cardiovasc Res 32: 764 –771, 1996. 10. The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 329: 977– 986, 1993. 11. Steiner DF: Evidence for a precursor in the biosynthesis of insulin. Trans NY Acad Sci 30: 60 –68, 1967. 12. Wahren J, Johansson BL, Wallberg-Henriksson H, et al: C-peptide revisited—new physiological effects and therapeutic implications. J Intern Med 240: 115–124, 1996. 626
13. Johansson BL, Sjo¨ berg S, and Wahren J: The influence of human C-peptide on renal function and glucose utilization in type I diabetic patients. Diabetologia 35: 121–128, 1992. 14. Sima AAF, Grunberger G, Jo¨ rnvall H, et al: Proinsulin C-peptide—a consensus statement. Int J Exp Diabetes Res 2: 145–151, 2001. 15. Sima AAF, Zhang W, Sugimoto K, et al: C-peptide prevents and improves chronic type I diabetic polyneuropathy in the BB/Wor rat. Diabetologia 44: 889 –897, 2001. 16. Li ZG, Zhang W, Grunberger G, et al: Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 946: 221– 231, 2002. 17. Pierson CR, Zhang W, and Sima AAF: Proinsulin Cpeptide replacement in type 1 diabetic BB/Wor-rats prevents deficits in nerve fiber regeneration. J Neuropath Exp Neurol 62: 765–779, 2003. 18. Rajasekaran M, Mondal D, Agrawal K, et al: Ex vivo expression of nitric oxide synthase isoforms and calmodulin in human penile cavernosal cells. J Urol 160: 2210 –2215, 1998. 19. Carmines PK, Fowler BC, and Bell PD: Segmentally distinct effects of depolarization on intracellular [Ca2⫹] in renal arterioles. Am J Physiol 265: F677–F685, 1993. 20. Grynkiewicz G, Poenie M, and Tsieu RY: A new generation of Ca2⫹ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440 –3450, 1985. 21. Flynn MD, Boolell M, Tooke JE, et al: The effect of insulin infusion on capillary blood flow in the diabetic neuropathic foot. Diabetes Med 9: 630 –634, 1992. ¨ stergren J, et al: The effects of 22. Tooke JE, Lins PE, O intravenous insulin infusion on skin microcirculatory flow in type 1 diabetes. Int J Microcirc Clin Exp 4: 69 –83, 1985. 23. Fostermann U, Closs EI, Pollock JS, et al: Nitric oxide synthase isozymes: characterization, purification, molecular cloning and functions. Hypertension 23: 1121–1131, 1994. 24. Moncada S, and Higgs A: The L-arginine-nitric oxide pathway. N Engl J Med 329: 2002–2012, 1993. 25. Forst T, DeLa Tour DD, Kunt T, et al: Effects of proinsulin C-peptide on nitric oxide, microvascular blood flow and erythrocyte Na⫹K⫹-ATPase activity in diabetes mellitus type 1. Clin Sci 98: 283–290, 2000. 26. Forst T, Kunt T, and Pohlmann T: Biological activity of C-peptide on the skin microcirculation in patients with insulin-dependent diabetes mellitus. J Clin Invest 101: 2036 –2041, 1998. 27. Jensen ME, and Messina EJ: C-peptide induces a concentration-dependent dilatation of skeletal muscle arterioles only in the presence of insulin. Am J Physiol 276: H1223– H1228, 1999. 28. Sima AAF: C-peptide and diabetic neuropathy. Expert Opin Drug Dev 12: 1471–1488, 2003. 29. Forst T, and Kunt T: Effects of C-peptide on microvascular blood flow and blood haemorheology. Exp Diabetes Res 5: 51–64, 2004. 30. Sessa WC: The nitric oxide synthase family of proteins. J Vasc Res 31: 131–143, 1994. 31. Ekberg K, Brismar T, Johansson BL, et al: Amelioration of sensory nerve dysfunction by C-peptide in patients with type 1 diabetes. Diabetes 52: 536 –541, 2003. 32. Li L, Oshida Y, Kusunoki M, et al: Rat C-peptide I and II stimulate glucose utilization in STZ-induced diabetic rats. Diabetologia 42: 958 –964, 1999. 33. MacNaul KL, and Hutchinson NI: Differential expression of iNOS and eNOS mRNA in human vascular smooth muscle cells and endothelial cells under normal and inflammation condition. Biochem Biophys Res Commun 196: 1330 – 1334, 1993. UROLOGY 64 (3), 2004
34. Rigler R, Pramanik A, Jonasson P, et al: Specific binding of proinsulin C-peptide to human cell membranes. Proc Natl Acad Sci USA 96: 13318 –13323, 1999. 35. Grunberger G, Qiang X, Li ZG, et al: Molecular basis for the insulinomimetic effects of C-peptide. Diabetologia 44: 1247–1257, 2001. 36. Grunberger G, and Sima AAF: The C-peptide signaling. Exp Diabetes Res 5: 25–36, 2004. 37. Li ZG, Qiang X, Sima AA, et al: C-peptide attenuates
UROLOGY 64 (3), 2004
protein tyrosine phosphatase activity and enhances glycogen synthesis in L6 myoblasts. Biochem Biophys Res Commun 280: 615–619, 2001. 38. Griffith OW, and Steehr DJ: Nitric oxide synthase: properties and catalytic mechanisms. Ann Rev Physiol 150: 73–77, 1995. 39. Hung A, Vernet D, Xie Y, et al: Expression of iNOS in smooth muscle cells from rat penile corpora cavernosa. J Androl 16: 469 –475, 1995.
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EFFECTS OF C-PEPTIDE ON EXPRESSION OF eNOS AND iNOS IN HUMAN CAVERNOSAL SMOOTH MUSCLE CELLS HAIKUN LI, LIPING XU, JOSEPH C. DUNBAR, CHIRPRIYA B. DHABUWALA, ANDERS A. F. SIMA
AND
ABSTRACT Objectives. To investigate the role of C-peptide alone or in conjunction with insulin on the expression of nitric oxide synthase (NOS) in human corpus cavernosum smooth muscle cells (HCSMCs). Erectile dysfunction, among diabetic patients, is a significant health problem. The specific causes of erectile dysfunction are unknown. It has been suggested that impairment of penile relaxation is related to a reduction of penile NOS. Plasma levels of C-peptide and insulin are decreased in individuals with type 1 diabetes and late-stage type 2 diabetes. Methods. Primary cultures were initiated from explants of HCSMCs. Confluent cells at passages 2 to 4 were assigned to one of four groups with the following incubation conditions: (a) 27 mM glucose, (b) 27 mM glucose and insulin, (c) 27 mM glucose and human recombinant (hr)C-peptide, and (d) 27 mM glucose, insulin, and hrC-peptide. After 24 hours, total RNA and protein were extracted from cells and subjected to reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. Intracellular Ca2⫹ was examined under the four conditions, using the Fura 2 method. Results. The least expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in HCSMCs was observed in cells exposed to 27 mM glucose alone. Increased expression of eNOS and iNOS was found after treatment with insulin or hrC-peptide alone, and the maximal expression of eNOS and iNOS was detected in HCSMCs exposed to both insulin and hrC-peptide. Western blot analyses using eNOS and iNOS antibodies confirmed the RNA data. These effects are likely mediated by the insulin-induced and/or C-peptide-induced increase in intracellular Ca2⫹. Conclusions. Our results demonstrated that C-peptide, in the presence of insulin, increases the expression of iNOS and eNOS in HCSMCs. These results suggest that C-peptide, especially in conjunction with insulin, may have beneficial effects on cavernosal smooth muscle relaxation. UROLOGY 64: 622–627, 2004. © 2004 Elsevier Inc.
E
rectile dysfunction (ED) is a significant health problem in the United States and develops in 35% to 75% of men with diabetes mellitus.1–3 Although ED is not life threatening, it has strong negative psychological effects on a man already burdened by diabetes. The specific causes of ED are unknown. The functional regulation of penile smooth muscle reThis study was supported in part by grants from the Juvenile Diabetes Research Foundation and the Thomas Foundation, Bloomfield Hills, Michigan to A. A. F. Sima. From the Departments of Urology, Physiology, Neurology, and Pathology, Wayne State University School of Medicine, Detroit, Michigan Reprint requests: Anders A. F. Sima, M.D., Ph.D., Department of Pathology, Wayne State University School of Medicine, Gordon H. Scott Hall, 540 East Canfield Avenue, Detroit, MI 48201 Submitted: June 10, 2003, accepted (with revisions): May 5, 2004
622
© 2004 ELSEVIER INC. ALL RIGHTS RESERVED
laxation is of prime importance for the development of normal erections. Among the neurotransmitters involved in cavernosal smooth muscle relaxation, nitric oxide (NO), synthesized by nitric oxide synthase (NOS), plays an essential role in ensuring normal penile erection.4 – 6 The potential sources of NO in the penis include neurons, sinusoidal endothelium, and corporeal smooth muscle cells.6 In diabetes, it has been suggested that impaired relaxation of blood vessels and the trabecular meshwork of smooth muscle corpora cavernosum is related to a reduction in penile NOS activity/expression, with resultant decreases in NO production.6 – 8 The discovery of insulin has greatly improved the metabolic control of diabetes; however, microvascular complications are still common among diabetic patients despite intensive insulin treatment 0090-4295/04/$30.00 doi:10.1016/j.urology.2004.05.005
and well-controlled glucose levels.9,10 C-peptide is a 31-amino acid peptide cleaved from pro-insulin and co-secreted with insulin.11 Plasma levels of Cpeptide and insulin are markedly decreased in individuals with type 1 diabetes and in those with the late stage of type 2 diabetes.12 Recent studies have demonstrated that C-peptide possesses biologic activities of its own.13–17 However, the role of C-peptide in the expression of penile NOS has not been studied. The present study was designed to test the hypotheses that C-peptide alone, or combined with insulin, influences the expression of NOS and that this is associated with intracellular Ca2⫹ regulation. First, we investigated the role of C-peptide in the expression of inducible NOS (iNOS) and endothelial NOS (eNOS) in human cavernosal smooth muscle cells (HCSMCs) in vitro. Second, we examined its regulatory role on intracellular Ca2⫹. MATERIAL AND METHODS CELL CULTURE AND TREATMENT HCSMCs were cultured as described by Rajasekaran et al.18 In brief, primary cultures were initiated using explants of human corpora cavernosa (2 mm3), obtained from nondiabetic patients receiving penile implants. The tissue was placed in Dulbecco’s growth media containing 20% fetal bovine serum for 5 days. The cells were then incubated with fresh Dulbecco’s growth media until confluence. The presence of smooth muscle cells was confirmed by smooth muscle actin staining. Monolayers of cells (80% to 90% confluence) at passages 2 to 4 were assigned to one of four groups incubated with (a) 27 mM glucose, (b) 27 mM glucose and insulin (500 U/mL, Sigma Chemical, St. Louis, Mo), (c) 27 mM glucose and human recombinant (hr)C-peptide (10 ng/mL, Schwarz Pharma GMBH, Monheim, Germany), or (d) 27 mM glucose, insulin, and hrC-peptide. After 24 hours of incubation, protein and total RNA was extracted and the expression of iNOS and eNOS detected by Western blotting and reverse transcriptase-polymerase chain reaction, respectively.
DETERMINATION OF NOS BY WESTERN BLOTTING Two NOS isoforms, iNOS and eNOS, were studied. Cells were homogenized in a lysis buffer (10 mM Tris-HCl, 1 mM ethylene-diamine-tetra-acetic acid or ethylenediaminetetraacetic acid, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktails). The homogenates were then centrifuged at 15,000g for 20 minutes at 4°C. Supernatants were collected, and the protein concentration was determined using a Bio-Rad protein assay kit. A total of 80 g protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. Equal loading of protein was ascertained by the Ponceau S stain. The membrane was then incubated with anti-iNOS or anti-eNOS antibody (Transduction Laboratories, San Diego, Calif) at a dilution of 1:2000 at 4°C overnight, and with a secondary antibody at a dilution of 1:2000 for 90 minutes. Immune complexes were detected with the enhanced chemiluminescence system (Amersham, Arlington Heights, Ill). The results were quantified with a scanning densitometer using an image analysis system with National Institutes of Health Image, version 1.55, software (Image Research, St. Catharines, Canada). UROLOGY 64 (3), 2004
DETERMINATION OF NOS BY REVERSE TRANSCRIPTASEPOLYMERASE CHAIN REACTION Total RNA was isolated from HCSMCs after 24 hours of treatment using an RNA isolation kit (Gibco, Gaithersburg, Md). cDNA was synthesized from 3 g total RNA with reverse transcriptase (SuperScript II, Gibco-BRL). Reverse transcription was performed at 42°C for 1 hour after incubation at 95°C for 5 minutes. cDNA amplification was performed according to the following temperature profile: 94°C for 1 minute; 60°C for 1 minute; and 72°C for 1.5 minutes. At the end of 35 cycles, the reaction was prolonged for 10 minutes at 72°C, and then 5 L was analyzed on a 1.5% agarose gel. Beta-actin was used as the control. Comparisons between the different conditions were made within the linear range of amplification of eNOS, iNOS, and beta-actin. The sequences of primers were 5⬘-GTGATGGCGAAGCGAGTGAAG-3⬘ and 5⬘-CCGAGCCCGAACACACAGAAC-3⬘ for eNOS; and 5⬘-TCCGAGGCAAACAGCACATCA-3⬘ and 5⬘-GGGTTGGGGGTGTGGTGATGT-3⬘ for iNOS; and 5⬘-AACCGCGAGAAGATGACCCAGATCATGTTT-3⬘ and 5⬘-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3⬘ for beta-actin.
MEASUREMENT OF INTRACELLULAR Ca2ⴙ Intracellular Ca2⫹ concentration was measured in cells exposed to normal (5 mM) or high (27 mM) glucose level with or without insulin (500 U/mL) or insulin plus hrC-peptide (10 ng/mL), using Fura 2 (TEF Labs, Austin, Tex) and dual excitation wavelength fluorescence microscopy.19,20 In brief, cells were incubated with physiologic saline solution containing 7 M Fura 2 acetoxymethyl ester, 0.09 g/dL dimethyl sulfoxide and 0.018 g/dL Pluronic F-127 (Molecular Probes, Eugene, Ore) for 60 minutes at 23°C. Cells were illuminated alternatively with excitation wavelengths of 340 and 380 nm. The emission wavelength was 510 nm. Background corrected data were collected using the Felix software package (Photon Technology International, Monmonth Junction, NJ).
STATISTICAL ANALYSIS
The data are presented as the mean ⫾ SEM. Statistical analysis was performed using analysis of variance followed by Newman-Keuls post hoc test. Probability values of less than 0.05 were considered statistically significant.
RESULTS EXPRESSION OF NOS mRNA Polymerase chain reaction analysis detected fragments of approximately 460 and 420 base pairs specific for iNOS and eNOS, respectively. The least expression of iNOS and eNOS mRNA was observed in HCSMCs treated with 27 mM glucose alone. A small increase in the levels of iNOS and eNOS mRNA occurred in cells treated either with insulin or hrC-peptide alone compared with that of 27 mM glucose alone. However, the maximal expression of eNOS and iNOS was detected in HCSMCs in the presence of both hrC-peptide and insulin (Fig. 1). EXPRESSION OF NOS PROTEIN Western blotting showed single bands of 130 kDa and 140 kDa for iNOS and eNOS, respectively. In cells treated with either hrC-peptide or insulin alone, a small increase was observed in the expression of these two isoforms compared with 27 mM 623
FIGURE 1. Expression of iNOS and eNOS mRNA in HCSMCs in primary culture. Lanes 1 to 4 correspond to cDNA fragments amplified from cells treated with 27 mM glucose, 27 mM glucose plus insulin, 27 mM glucose plus hrC-peptide, and 27 mM glucose plus insulin and hrC-peptide, respectively. Data shown from representative experiment of three independent experiments. FIGURE 3. Expression of eNOS protein in HCSMCs in primary culture. (A) Western blot analysis of eNOS expression in HCSMCs. Lanes 1 to 4 correspond to eNOS expression from cells treated with 27 mM glucose (basal), 27 mM glucose plus insulin, 27 mM glucose plus hrC-peptide, or 27 mM glucose plus insulin (Ins) and hrC-peptide, respectively. Data shown from representative experiment of three independent experiments. (B) Quantitative results using densitometric scanner. Data expressed as percentage of basal levels (27 mM glucose) for three independent experiments. *P ⬍ 0.01, **P ⬍ 0.001 versus basal condition; †P ⬍ 0.05 versus insulin or hrC-peptide alone.
FIGURE 2. Expression of iNOS protein in HCSMCs in primary culture. (A) Western blot analysis of iNOS expression in HCSMCs. Lanes 1 to 4 correspond to iNOS expression from cells treated with 27 mM glucose (basal), 27 mM glucose plus insulin, 27 mM glucose plus hrC-peptide, or 27 mM glucose plus insulin (Ins) and hrC-peptide, respectively. Data shown from representative experiment of three independent experiments. (B) Quantitative results using densitometric scanner. Data expressed as percentage of basal levels (27 mM glucose) for three independent experiments. *P ⬍ 0.01; **P ⬍ 0.001 versus basal condition; †P ⬍ 0.005 versus insulin or hrC-peptide alone.
glucose alone. The maximal expression of the two isoforms was detected in cells treated with both hrC-peptide and insulin (Fig. 2A and 3A). Quantitative densitometry from three independent experiments revealed that the level of iNOS in HCSMCs increased by 1.4-fold (P ⬍ 0.05), 1.2-fold (P ⬍ 0.05) and 2.3-fold (P ⬍ 0.001) above basal levels in cells treated with insulin alone, hrC-peptide alone, and both hrC-peptide and insulin, respectively (Fig. 2B). The expression of eNOS was increased by 1.3-fold (P ⬍ 0.05), 1.2-fold (P ⬍ 0.05), and 1.6-fold (P ⬍ 0.001) above the basal levels in cells treated with insulin alone, hrC-pep624
tide alone, and both hrC-peptide and insulin, respectively (Fig. 3B). INTRACELLULAR Ca2ⴙ No differences in intracellular Ca2⫹ were detected between cells incubated with 5 and 27 mM glucose. The addition of insulin alone (500 U/ mL) increased intracellular Ca2⫹ in cells exposed to both glucose concentrations (P ⬍ 0.01 for both). When incubated with both insulin and hrC-peptide (10 ng/mL), a small additional increase (P ⬍ 0.05) in intracellular Ca2⫹ was seen in cells exposed to 5 mM glucose; however, the intracellular Ca2⫹ expression was maximal (P ⬍ 0.001 versus control) in cells exposed to 27 mM glucose and insulin plus hrC-peptide (Fig. 4). COMMENT The present data have demonstrated that C-peptide increases the expression of iNOS and eNOS in HCSMCs. This was further increased in the presence of both C-peptide and insulin, suggesting a synergistic effect on the regulation of NO in HCSMCs. The vasodilatory effect of insulin21,22 has been linked to the release of NO from endothelial cells and to decreased NO production in insulinUROLOGY 64 (3), 2004
FIGURE 4. Intracellular Ca2⫹ concentrations in HCSMCs incubated in either 5 or 27 mM glucose and with 500 U/mL insulin or insulin and 10 ng/mL hrC-peptide. *P ⬍ 0.01; **P ⬍ 0.001 versus respective concentration of glucose alone; †P ⬍ 0.005 versus 27 mM glucose plus insulin and hrC-peptide.
deficient diabetes,23,24 in agreement with the present data. The additional enhancing effect of C-peptide on NO synthesis in the presence of insulin is in keeping with recent clinical and experimental data.25–28 The present data suggest that the potentiating effect of C-peptide on NO synthesis is mediated by an increase in Ca2⫹ influx, also consistent with recent findings.29 The functional regulation of penile smooth muscle relaxation is of prime importance for the development of normal erections. Of the various mediators involved in cavernosal smooth muscle relaxation, NO plays an essential role in ensuring normal penile erections.4 – 6 NO is synthesized by NOS in a reaction that converts arginine and oxygen into citrulline and NO.5 Measurement of NO production has been accomplished by examining the enzyme responsible for NO synthesis (NOS). To date, several isoforms of NOS have been purified and cloned.30 The inducible isoform of NOS (iNOS) is associated primarily with macrophage function and is activated by specific cytokines. The endothelial (eNOS) and neuronal isoforms of NOS are constitutive and are activated, in part, by increased concentrations of intracellular calcium and calmodulin binding. In this study we have demonstrated that the expression of iNOS and eNOS is increased in HCSMCs after 24 hours of treatment with C-peptide, especially in the presence of insulin. These findings suggest that C-peptide may have a beneficial effect on impaired erectile responses. However, the evidence for a direct link between C-peptide deficiency and ED needs to be determined in clinical trials. UROLOGY 64 (3), 2004
C-peptide is a 31-amino acid peptide cleaved from pro-insulin and co-secreted with insulin.11,28 Plasma levels of C-peptide and insulin are markedly decreased in individuals with type 1 diabetes, as well as in those with late-stage type 2 diabetes.12,28 C-peptide was initially believed to have no biologic effects apart from its role in insulin biosynthesis. However, recent findings of the physiologic effects of C-peptide and beneficial effects of C-peptide replacement in patients with type 1 diabetes have prompted renewed interest. Studies have indicated that in patients with type 1 diabetes who lack endogenous C-peptide, the administration of C-peptide improves renal function, stimulates muscle microcirculation and glucose use, improves blood-retinal barrier function, and improves sensory nerve conduction velocity.12–14,31 The mechanism underlying the observed effects of C-peptide is not clear. Previous studies have suggested that C-peptide, by an increase in intracellular Ca2⫹, stimulates eNOS activity,28,29,32 which is in agreement with the present findings showing increased intracellular Ca2⫹ in cells exposed to both insulin and C-peptide. The subcellular mechanism for C-peptide’s ability to stimulate iNOS expression remains unclear. MacNaul and Hutchinson33 have suggested that iNOS may have negative feedback effects on the eNOS. Our findings did not support this notion, because both iNOS and eNOS expression was increased by Cpeptide and increased further when exposed to both hormones, suggesting that C-peptide acts synergistically with insulin. Evidence for specific binding of C-peptide to a cell surface receptor has been demonstrated. It has been suggested by Rigler et al.34 that this is a G protein-coupled receptor. More recent studies have not been able to confirm this, but have suggested that C-peptide may bind to the insulin receptor itself at a different ligand site.35,36 The latter is consistent with C-peptide signaling by way of the insulin signaling pathways and its enhancing effect on insulin signaling intermediaries in the presence of insulin.35–37 eNOS has been identified in a variety of cell types in addition to the endothelial cell.38 Hung et al.39 reported that rat cavernosal smooth muscle cells express a Ca2⫹-calmodulin dependent constitutive form of NOS (eNOS) in culture. The results from Rajasekaran et al.18 have also shown that HCSMCs in culture express both eNOS and iNOS. In our study, we have demonstrated that HCSMCs, when treated with C-peptide alone or in conjunction with insulin, express both eNOS and iNOS in culture. These observations suggest that cavernosal smooth muscle cells have the ability to synthesize their own NO. Although unlikely, it cannot be totally excluded that this could potentially be induced by in vitro conditions. Thus, C-peptide may 625
exert its function through NO-mediated mechanisms in these cells. Additional support for the idea that C-peptide’s effects may be mediated by a NOmediated pathway is provided by the observation that C-peptide induces a concentration-dependent dilation of rat skeletal muscle arterioles mediated by NO.27 Moreover, C-peptide also stimulate eNOS, with release of NO in a concentration-dependent manner from aortic endothelial cells, effects that are abolished by NOS inhibitors.32 CONCLUSIONS We have demonstrated that C-peptide, in the presence of insulin, increases the expression of iNOS and eNOS in HCSMCs, probably by way of a Ca2⫹-mediated mechanism. This suggests that Cpeptide, especially in conjunction with insulin, may play a regulatory role through NO-mediated mechanisms on HCSMCs, and that its replacement in patients with insulin-dependent diabetes may have therapeutic effects. ACKNOWLEDGMENT. To Dr. Suresh Sikka for providing the human cavernosal smooth muscle cells. REFERENCES 1. Krane RJ, Goldstein I, and Saenz De Tejada I: Impotence. N Engl J Med 321: 1648 –1659, 1989. 2. Kolodny RC, Kahn CB, Goldstein HH, et al: Sexual dysfunction in diabetic men. Diabetes 23: 306 –309, 1974. 3. Feldman HA, Goldstein I, Hatzichristou DG, et al: Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol 151: 54 –61, 1994. 4. Trigo-Rocha F, Hsu GL, Donatucci CF, et al: The role of cyclic adenosine monophosphate, cyclic guanosine monophosphate, endothelium and nonadrenergic, noncholinergic, neurotransmission in canine penile erection. J Urol 149: 872– 877, 1993. 5. Lowenstein CJ, Dinerman JL, and Snyder SH: Nitric oxide: a physiologic messenger. Ann Intern Med 120: 227– 237, 1994. 6. Burnett AL: Nitric oxide in the penis: physiology and pathology. J Urol 157: 320 –324, 1997. 7. Saenz de Tejada I, Goldstein I, Azadzoi K, et al: Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men impotence. N Engl J Med 320: 1025–1030, 1989. 8. Seftel AD, Vaziri ND, Ni Z, et al: Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through iNOS or eNOS. Urology 50: 1016 –1026, 1997. 9. Tooke JE: Microvasculature in diabetes. Cardiovasc Res 32: 764 –771, 1996. 10. The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 329: 977– 986, 1993. 11. Steiner DF: Evidence for a precursor in the biosynthesis of insulin. Trans NY Acad Sci 30: 60 –68, 1967. 12. Wahren J, Johansson BL, Wallberg-Henriksson H, et al: C-peptide revisited—new physiological effects and therapeutic implications. J Intern Med 240: 115–124, 1996. 626
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