CC BY
19
RegpLatory Mechanisms
in Biosystems
Regulatory Mechanisms
in Biosystems
ISSN 2519-8521 (Print) ISSN 2520-2588 (Online) Regul. Mech. Biosyst., 8(2), 287-292 doi: 10.15421/021745
Protein kinase C (PKC) involved in enhancement of a1-adrenoceptor-mediated responses of the main pulmonary artery in rats with diabetes mellitus
I. V. Kizub*, O. I. Kharchenko*, O. S. Kostiuk*, L. I. Ostapchenko*, A. I. Soloviev**
*Taras Shevchenko National University of Kyiv, Kyiv, Ukraine
**lnstitute of Pharmacology and Toxicology of National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
Article info
Received 24.03.2017 Received in revised form
20.04.2017 Accepted 23.04.2017
Taras Shevchenko National University of Kyiv, Academician Glushkov Ave., 2, Kyiv, 03022, Ukraine. Tel. +38-044-456-02-88. E-mail: buzzmann@ukr. net, 77olgaz@gmail. com, [email protected], [email protected] tonysolpharm@gmail. com.
Kizub, I V., Kharchenko, O. I, Kostiuk, O. S., Ostapchenko, L I., & Soloviev, A I (2017). Protein kinase C (PKC) involved in enhancement of a1-adrenoceptor-mediated responses of the main pulmonary artery in rats with diabetes mellitus. Regulatory Mechanisms in Biosystems, 8(2), 287-292. doi:10.15421/021745
Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Moreover, DM can strongly affect pulmonary circulation, enhancing the wall thickness of the pulmonary arteries, changing their tone and contractility, and gas exchange in the lungs. It can lead to marked loss of lung function and respiratory efficiency. It is also known that protein kinase C (PKC) activity increases in DM and that PKC is involved in the mechanisms of DM-associated vascular complications. However, the effect of DM on pulmonary artery tone has been poorly investigated and the role of PKC in this remains unknown. The aim of this study was to investigate changes in contractility of pulmonary arteries in rats with DM and to determine the possible role of PKC in this process. Experimental type 1 DM was elicited in male Wistar rats by single streptozotocin (STZ, 65 mg/kg) injection. DM was verified by the presence of hyperglycaemia. The investigation was performed on the isolated rings of the main pulmonary arteries using the method of vascular tone registration. Phenylephrine (PhE, 0.1 nM - 1 mM) caused dose-dependent constriction of the pulmonary arteries. The pD2 (negative logarithm of the agonist concentration required for half-maximum response) of this constriction increased in rats with DM, however significant changes in amplitude of PhE-induced constriction were not observed. PKC inhibition with chelerythrine and staurosporine (1 |rM) significantly shifted PhE the concentration-response curve to the right in intact diabetic vessels but had no effect on sensitivity to PhE in deendothelised diabetic vessels. Our data suggest that type 1 DM leads to enhancement in pulmonary artery a1-adrenoceptor-mediated contractility and PKC activity in the endothelium rather than in vascular SMCs is involved in this process.
Keywords: diabetes mellitus; protein kinase C; vascular tone; vascular smooth muscle; endothelium; pulmonary artery
pathways (Kizub et al., 2014). PKC is a family of regulatory enzymes (serine/threonine kinases) (Cosentino-Gomes et al., 2012) that plays a prominent role in the signal transduction of several vascular functions including regulation of vascular smooth muscle contractility (Somlyo and Somlyo, 2003; Cosentino-Gomes et al., 2012). It has been shown that dysfunctions of these systems are associated with the diabetic state and involve PKC-dependent mechanisms which are implicated as an important players in the pathogenesis of diabetic microangio-pathy (Clarke and Dodson, 2007; Kizub et al., 2014) and macroangio-pathy (Geraldes and King, 2010; Kizub et al., 2014). Numerous PKC isozymes (a, Y, s, H, C, 8 and t/X) have been shown to be
activated or overexpressed in vascular smooth muscle cells (SMCs) and the endothelium of different vascular regions in subjects with diabetes (Ramana et al., 2005; Klymenko et al., 2014).
Despite a number of studies addressed to vascular complications in DM, there is little known about the effect of DM on pulmonary circulation. The present study has been devoted to an investigation of alterations in pulmonary artery tone associated with DM and the possible role of PKC in this process.
Material and methods
Experiments were performed on isolated vascular rings obtained from the main pulmonary artery of male Wistar rats
Introduction
Diabetes mellitus (DM) is a complex syndrome that is rapidly rising in incidence throughout the world. Hyperglycemia and alterations in metabolism are the most severe components of DM (Zimmet, 2011). The adverse long-term effects of DM involve many organ systems and are associated with a complex pathology leading to a large number of secondary cellular and subcellular changes.
DM leads to multiple dysfunctions including cardiovascular diseases, one of the major causes of morbidity, mortality, end-stage renal disease, and blindness (Madonna and de Caterina, 2011). The macrovascular manifestations of DM include angiopathy, atherosclerosis, medial calcification, and arterial hypertension mostly located in the coronary and carotid arteries (Whiteley et al., 2005; Cosson et al., 2006), cerebral vessels, and the large peripheral arteries of the lower extremities (Funk et al., 2012). Increased blood flow and vascular tone elevation have been documented in diabetes (Madonna and de Caterina, 2011).
Hyperglycaemia is a key factor responsible for the development of vascular complications in diabetes (Boussageon et al., 2011; Madonna and de Caterina, 2011). Several hyperglycaemia-associa-ted mechanisms have been identified as contributing to the development of vascular dysfunction associated with DM. One of such mechanisms may involve activation of protein kinase C (PKC)
weighing 190-210 g. All experimental procedures conformed to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and were approved by the Ethics Committee of the Educational and Scientific Centre "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, Ukraine and State Institution "Institute of Pharmacology and Toxicology of NAMS of Ukraine", Kyiv, Ukraine. Two groups of animals were used: diabetic rats injected with streptozotocin (STZ, 60-65 mg/kg, i.p.) and maintained for 9 weeks, and age-matched controls. STZ was dissolved in buffer solution containing 0.9% NaCl and 10 mM citrate (pH = 4.6). DM development was verified by the presence of hyperglycaemia (plasma glucose higher than 20 mM) every 4 weeks after streptozo-tocin injection, and on the day of experimentation. The diabetic group of animals (blood glucose 26.02 ± 5.04 mM, n = 8) was compared with the control rats (blood glucose 6.74 ± 0.12 mM, n = 8; two-tailed t-test P < 0.05). Blood samples were obtained from the tail tip and analyzed using a glucose meter Bionime Rightest GS300 (Bionime Gmbh, Switzerland).
The animals were killed by cervical dislocation. The heart and lungs were rapidly removed and placed into Krebs' physiological salt solution (composition in mM: 133.00 NaCl, 16.30 NaHCO3, 1.05 MgCl2, 1.38 NaH2PO4, 4.70 KCl, 7.00 D-glucose, 2.75 CaCl2 and 10 HEPES, pH 7.4). For rings from diabetic animals, the concentration of glucose was increased to 25 mM. Vessels were dissected free, cleaned of connective and adipose tissue, cut into 23 mm rings and mounted on fine stainless steel hooks in organ baths perfused with warmed to 37 °C Krebs' solution. For vascular tone measurement capacitive tension detectors (Danish Myo Technology, Aarhus, Denmark) and LabScribe 2 (World Precession Instrument Inc., USA) software were used. The resting tension of the vessels was gradually increased to 0.7-0.8 g. After 1 hour equilibration, each vascular ring was exposed twice to 60 mM KCl exposure to assess its viability and reproducibility of contractions.
In experiments with deendothlised vessels the endothelium was disrupted chemically with 15 min incubation of the vascular rings in Krebs' solution containing 0.1 mg/ml of saponine. Successful endothelium removal was determined by absence of 10 pM acetylchole-induced relaxation following the contraction in response to 1 mM phenylephrine (PhE) application.
Data are presented as mean ± SE with n indicating the number of vascular preparations tested. Contractile responses were expressed as a percentage of the maximum response produced by 60 mM KCl or 1 mM PhE. The sensitivity to the agonist was expressed as pD2 (negative logarithm of the agonist concentration required for half-maximum response). In order to determine this parameter, data points were calculated using the Origin 8.1 software (OriginLab Corporation, Northampton, MA, USA) with the 'DoseResp' function in the following form:
100
T =-,
1 +10 L°g (x0~x)'p
where T is normalized tension (expressed as % of maximal contraction) at the agonist concentration x (expressed as negative logarithm), x0 is the midpoint of the concentration-effect curve (e.g. T = 50%) and p is the slope factor of the curve. Mean values were obtained by averaging pD2 values obtained from individual preparations. Statistical comparisons were made using Student's unpaired two-tailed t-test for two groups. Differences for P < 0.05 were considered to be statistically significant.
Results
Effect of DM on intact pulmonary artery contractility. In control intact pulmonary artery rings isolated from non-diabetic rats, phenylephrine (PhE), a selective aradrenoceptor agonist, applied at ascending concentrations (0.1 nM - 1 mM) evoked concentration-dependent contractions with a maximal response (Emax) 146.76 ± 18.92 % (n = 16) of constriction evoked by 5-minute application of
60 mM KCl (Fig. 1 and 2A). A plateau response was obtained after the addition of each test PhE concentration before the addition of a subsequent dose. pD2 value of PhE in control vessels consisted 7.49 ± 0.19 (n = 16) (Fig. 2B).
In intact pulmonary arteries isolated from diabetic rats, the responsiveness to PhE was enhanced since the diabetic vessels responded to lower concentrations of the agonist. pD2 value of PhE in diabetic vessels was 8.02 ± 0.09 (n = 16, P < 0.05) (Fig. 2). In contrast, pulmonary artery rings from diabetic animals showed no significant difference in Emax between nondiabetic control and diabetic rats. It was 130.4 ± 10.5% (n = 16; P > 0.05) of constriction evoked by 60 mM KCl (Fig. 1).
Fig. 1. Mean data of relevant amplitude of the contractile responses to phenylephrine (PhE, 0.1 nM - 1 mM) in intact (E+) pulmonary artery from control (Ctrl) and diabetic (DM) rats before and after PKC inhibition with chelerythrine (Chel, 1 ^M) and staurosporine (Stsp, 1 ^M); * - P < 0.05
Effect of DM on deendothelised pulmonary artery contractility. To evaluate the role of the endothelium in pulmonary artery contractility in DM experiments were performed on deendothelised vascular rings. In deendothelised pulmonary arteries from control rats, 0.1 nM - 1 mM PhE application led to vasoconstriction development with a maximal amplitude of 99.4 ± 22.5% (n = 8) of contraction evoked by 60 mM KCl (Fig. 3). pD2 value of PhE-induced constriction in these vessels was 7.55 ± 0.16 (n = 8) (Fig. 4).
In the deendothelised pulmonary artery of animals with experimental DM, the dose-response curve of PhE was significantly shifted to the right as compared to the intact vessels of the control animals, showing decrease in vascular sensitivity to the agonist with pD2 value of 7.09 ± 0.22 (n = 8, P < 0.05) (Fig. 4). In contrast to deendothelised control vessels, significant changes in the amplitude of PhE-evoked contraction in diabetic pulmonary artery rings were not observed (138.7 ± 24.0%; n = 8; P > 0.05; Fig. 3).
Effect of PKC inhibition on intact pulmonary artery contractility in norm and DM. To investigate the role of PKC in pulmonary artery tone alterations in DM, vascular tissues were pre-treated for 20 min with combination of potent cell-permeable PKC inhibitors chelerythrine (1 ^M) and staurosporine (1 ^M). PKC inhibition had no significant effect on the amplitude of Phe-induced constriction in intact pulmonary arteries of control rats. Maximum contraction in response to 100 ^M PhE was 175.5 ± 20.8% (n = 12; P > 0.05 as compared to control) (Fig. 1). On the other hand, PKC inhibition resulted in an unexpected leftward shift of the concentration-response curve for PhE with pD2 value 8.55 ± 0.49 (n = 8; P < 0.05) (Fig. 2).
In diabetic pulmonary arteries PKC inhibition significantly shifted the concentration-response curve for PhE to the right (Fig. 2). Mean pD2 value in diabetic arteries was 6.67 ± 0.29 (n = 12;
P < 0.05) compared with the control tissue. PKC inhibition also significantly decreased amplitude of PhE-induced constriction in pulmonary arteries from diabetic animals (Fig. 2). In these conditions its maximum was 28.3 ± 8.6% (n = 12; P < 0.05) of constriction evoked by 60 mM KCl (Fig. 1).
Fig. 2. The changes in sensitivity to phenylephrine (PhE) in intact (E+) pulmonary artery from control and diabetic rats: A - original traces illustrating agonist concentration-response in intact control (top) and diabetic (bottom) rat pulmonary artery preparations; B - mean data of normalized amplitude of the contractile responses plotted versus PhE concentration (0.1 nM - 1 mM) in intact (E+) pulmonary artery from control (Ctrl) and diabetic (DM) rats before
and after PKC inhibition with chelerythrine (Chel, 1 ^M) and staurosporine (Stsp, 1 ^M); vertical dotted lines represent pD2 value of PhE-induced constriction dose-response curves fitted by equation 1 (see Methods section); it should be noted that dose-response curves "DM" and "Ctrl + Chel + Stsp" are superposed; * - P < 0.05
Effect of PKC inhibition on deendothelised pulmonary artery contractility in norm and DM. PKC inhibition with chelerythrine and staurosporine had no effect on Emax of deendothelised control pulmonary artery contraction evoked by 100 ^M PhE with value 64.2 ± 14.9% (n = 9; P > 0.05) (Fig. 3). The sensitivity of deendothelised vessels from control animals to PhE also was not changed, and pD2 value of PhE-induced constriction in these vessels was 7.77 ± 0.31 (n = 9; P > 0.05) (Fig. 4).
In diabetic endothelium-free vascular tissues PKC inhibition also had no effect on maximal amplitude of PhE-induced vasoconstriction. It was 103.0 ± 27.3% (n = 8; P > 0.05) (Fig. 3). PKC inhibition also did not affect concentration-response curve for PhE in deendothelised vessels from diabetic animals. pD2 value of PhE-induced constriction in these vessels was 6.99 ± 0.32 (n = 8; P > 0.05) as compared to control (Fig. 4). Table 1 represents comparison of pD2 values which characterize PhE-induced constriction dose-response curve in both intact and deendothelised pulmonary arteries from control and diabetic rats.
Fig. 3. Mean data of relevant amplitude of the contractile responses
to phenylephrine (PhE, 0.1 nM - 1 mM) in deendothelised (E-) pulmonary artery from control (Ctrl) and diabetic (DM) rats before and after PKC inhibition with chelerythrine (Chel, 1 ^M) and staurosporine (Stsp, 1 ^M); * - Р < 0.05
Fig. 4. Mean data of normalized amplitude of the contractile responses plotted versus phenylephrine (PhE) concentration (0.1 nM -1 mM) reflecting the changes in sensitivity to PhE in deendothelised
(E-) pulmonary artery from control (Ctrl) and diabetic (DM) rats before and after PKC inhibition with chelerythrine (Chel, 1 ^M) and staurosporine (Stsp, 1 ^M); vertical dotted lines represent pD2 value of PhE-induced constriction dose-response curves fitted by equation 1 (see Methods section); it should be noted that dose-response curves for control and DM are superposed;* - Р < 0.05
Table 1
Mean pD2 for PhE-induced constriction dose-response curve in intact (Е+) and deendothelised (Е-) pulmonary artery from control and diabetic (DM) rats in the absence or presence of combined action of chelerythrine (1 ^M) and staurosporine (1 ^M)
Control
Control + chelerythrine and staurosporine
DM
DM + chelerythrine and staurosporine
Е+
pD2-
7.49 ± 0.19 (n = 16)
8.55 ± 0.49 (n = 8)*
8.02 ± 0.09 (n = 16)*
6.67 ± 0.29 (n = 12)*
7.55 ± 0.16 (n = 8)
7.77 ± 0.31 (n = 9)#
7.09 ± 0.22 (n = 8)*#
6.99 ± 0.32 (n = 8)
Note: * - P < 0.05 in comparison with pulmonary artery rings from control animals; # - P < 0.05 in comparison between E+ and E- pulmonary artery rings.
Discussion
The data obtained clearly indicate that type 1 DM development leads to increase in sensitivity to PhE in rats' pulmonary arteries reflecting enhancement in vascular a1-adrenoceptor-mediated contractility. However, PKC inhibition in deendothelised vessels from
Е
diabetic animals had no effect on the sensitivity to PhE. It may suggest that mechanisms of pulmonary artery contractility enhancement in DM are associated with activity of PKC in the endothelial cells rather than in vascular SMC. There is a variety of evidence to indicate that sensitivity to a1-adrenoceptors-mediated stimulation is markedly elevated in STZ-diabetic rats' systemic arteries: tail artery (Kizub et al., 2010), mesenteric artery (White and Carrier, 1990; Mueed et al., 2005; Kizub et al., 2010), and aorta (Xavier et al., 2003) whereas other studies have shown an increase in amplitude of a1-adrenoceptors-mediated vasoconstriction in these arteries (Abebe and McLeod, 1991; Chow et al., 2001; Lee et al., 2011). Similar results showing enhanced noradrenaline-induced vasoconstriction have been obtained in high glucose concentration in the aorta of OLETF (Otsuka Long-Evans Tokushima Fatty) rat, an experimental model of insulin-independent DM (Nobe et al., 2003). On the other hand, a few studies have shown that the aortas of STZ-diabetic rats exhibited no changes in sensitivity to PhE (Chang and Stevens, 1992; Kizub et al., 2010).
In contrast to our data, Gurney and coauthors have shown that STZ-induced diabetes significantly blunted the maximum response of rats' conduits, but not resistance pulmonary arteries to PhE, without changes in the sensitivity to PhE. Endothelium-dependent vasodilatation of these vessels was also unaffected in DM (Gurney and Howarth, 2009). Other researchers have demonstrated that in the intrapulmonary arteries of STZ-diabetic rats the concentration-response curve PhE was characterized by an increased maximal response in the diabetic group compared with the control one without changes in pD2 value. Endothelium-dependent relaxant response was dramatically reduced in pulmonary arteries from diabetic rats (Lopez-Lopez et al., 2008).
Our data indicate that PKC activity is involved in elevated ar adrenoceptors-mediated vasoconstriction in the pulmonary arteries of STZ-diabetic rats. It has been shown by us previously that PKC inhibition suppressed elevated sensitivity to PhE in the smooth muscle of the tail artery of STZ-diabetic rats (Kizub et al., 2010). In our present study two potent inhibitors of PKC, chelerythrine and staurosporine were both used to achieve complete inhibition of PKC. Chelerythrine is a specific inhibitor of PKC substrate-binding site (Herbert et al., 1990), whereas staurosporine (or antibiotic AM-2282) is a selective inhibitor of ATP-binding site of PKC (Ishii et al., 1996). We have previously shown that changes in sensitivity of systemic vessels (aorta and tail artery) to aradrenoceptors agonists in PKC inhibition in healthy rats differ. It has been demonstrated that PKC inhibition had no significant effect on the contractile responses to PhE of the tail artery of healthy rats but evoked decrease in sensitivity to PhE in aorta (Kizub et al., 2010). On the other hand, there is evidence that PKC inhibition with other potent PKC inhibitors Ro-318220 and calfostin C in mesenteric arteries of healthy rats had no effect on maximum amplitude of norepinephrine (NE)-evoked contraction but significantly elevated its sensitivity to NE (Mueed et al., 2005).
The role of PKC in enhancement in contractility of the pulmonary artery has not been demonstrated before and is clearly shown in the present study for the first time. It has only been demonstrated previously by Yamada and Yokota (Yamada and Yokota, 1997) that in human pulmonary arteries endothelial cell culture PKC activators stimulated endothelin-1 (ET-1) release, whereas PKC inhibition with staurosporine led to decrease in both basal and stimulated levels of ET-1. These authors have also demonstrated that PKC activators stimulated prostacyclin (or prostaglandin I2, PGI2) release (Yamada and Yokota, 1997).
Although it has been shown in systemic vessels (aorta, mesenteric and tail artery) from STZ-diabetic rats that PKC inhibition with chelerythrine had no effect on sensitivity of these arteries to PhE (Kizub et al., 2010), studies on the deendothelised mesenteric artery of diabetic rats have demonstrated that PKC inhibition suppressed elevation in NE-induced contractile response (Mueed et al., 2005). In contrast to this, other authors have shown that PKC inhibition did not affect hyperreactivity to prostaglandin
E2 (PGE2) in the mesenteric artery of Goto-Kakizaki rats with 2-type DM (Ishida et al., 2012).
Our results showed that PKC inhibition in the deendothelised pulmonary artery from diabetic animals had no effect on sensitivity to PhE. It may allow us to suggest that mechanisms of pulmonary artery contractility enhancement in DM can be associated with activity of PKC in the endothelium. PKC involvement in vascular contractile abnormalities in DM can be mediated by a few mechanisms described. PKC activation in vascular endothelium in DM can result in endothelium-dependent vasodilator dysfunction via inhibition of the pathways associated with nitric oxide (NO) (Ishii et al., 1996; Beckman et al., 2002; Pitocco et al., 2010; Kizub et al., 2014), endothelium-derived hyperpolarizing factor (EDHF) (Gao et al., 2011), and PGI2 (Cosentino et al., 2003). Secondly, PKC activation in the endothelium in DM can enhance endothelium-dependent vasoconstriction mediated by ET-1 (Matsumoto et al., 2009), PGE2 and thromboxane A2 (TXA2) (Cosentino et al., 2003). Activation of nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidase or Nox) have also been shown to be involved in PKC-mediated endothelial dysfunction in DM via reactive oxygen species (ROS) formation (Gao et al., 2011; Kolluru et al., 2012; Kizub et al., 2014).
NO-associated mechanisms underlying diabetes-associated vascular dysfunction may include decreased endothelial nitric oxide synthase (eNOS) activity and expression (Hirata et al., 1995), uncoupling of eNOS and degradation of NO secondary to enhanced superoxide production (Cosentino et al., 2003; Pitocco et al., 2010), attenuation of NO signaling and decreased NO bioavailability (Matsumoto et al., 2009). In diabetes PKC may affect NO bioavailability not only via intracellular accumulation of ROS (Pitocco et al., 2010) but also by decreasing eNOS activity (Hirata et al., 1995). It is established that dysfunction of eNOS in DM can be associated with its suppression by some PKC isoforms (Hirata et al., 1995; Ishii et al., 1996; Bohlen and Nase, 2001; Mehta et al., 2009). As has been shown in DM, PKC can phosphorylate eNOS (Hirata et al., 1995) on its inhibitory site Thr495 (Fleming et al., 2001) and reduces eNOS phosphorylation on the activating Ser1177 site (Michell et al., 2001) blunting eNOS activity. On the other hand, PKC-mediated inhibition in eNOS activity is linked to the ability of PKC to phosphorylate another inhibitory phosphorylation site Thr497 of eNOS reducing its affinity for calmodulin and, hence, the generation of NO (Matsubara et al., 2003). Alternatively, PKC-dependent reduction in eNOS expression has been shown in retinal (Suzuma et al., 2002) and aortic (Hink et al., 2001) endothelial cells in diabetes. Decreased eNOS expression in diabetes can occur through PKC-mediated activation of vascular Nox by inducing ROS-dependent scavenging and reducing NO level (Inoguchi et al., 2000).
EDHF also can be involved in endothelial dysfunction development in DM. It has been established that EDHF-type vasorelaxation is impaired in DM (Matsumoto et al., 2006; Gao et al., 2011; Leo et al., 2011). The relaxant pathway associated with SMCs hyperpolarization is thought to be independent of NO and prostacyclin production by the endothelial cells and has been attributed to the release of EDHF (Feletou and Vanhoutte, 2010). The identity of EDHF remains controversial, but rather than a true chemical mediator of endothelium-dependent hyperpolarization, there is considerable support for the view that EDHF reflects a hyperpolarization signal that is mediated from endothelial cells to vascular SMCs via myoendothelial gap junctions (MEGJs) (Matsumoto et al., 2006; Figueroa and Duling, 2009; Feletou and Vanhoutte, 2010). Direct communication within and between endothelial cells and SMCs through gap junctions (GJs) is an important modulator of vascular tone and essential in the control and coordination of vascular function (Figueroa and Duling, 2009; Feletou and Vanhoutte, 2010). The vascular GJs are composed of intercellular channels clusters allowing the direct passage of electrical current and small signaling molecules between adjacent cells (Figue-roa and Duling, 2009). In vascular tissues GJs consist of connexin proteins (Cx37, Cx40, Cx43 and Cx45) (Brisset et al., 2009).
Connexin phosphorylation is highly sensitive to glucose concentration through the regulation via PKC dependent signaling pathway (Lin and Takemoto, 2005). It has been demonstrated that diabetes affects MEGJs function in resistance arteries (Lin and Takemoto, 2005; Georgescu et al., 2006). Endothelial Cx37 and Cx40 protein expression levels and endothelial GJs consisting of these connexins have been reported to be reduced in different vascular regions of STZ-induced type 1 diabetic mice (Hou et al., 2008; Makino et al., 2008). A few studies have reported that exposure to high glucose results in a down-regulation of Cx43 in vascular endothelial cells (Sato et al., 2002; Chen et al., 2008; Li and Roy, 2009).
Inoguchi and coauthors have reported in bovine aortic endothelial cells, that hyperglycemia inhibited gap junctional intercellular communication via PKC (Inoguchi et al., 1995; Inoguchi et al., 2001). Similar results have been demonstrated in retinal microvessels of rats with STZ-induced diabetes (Oku et al., 2001). It has been also demonstrated that high glucose levels, via PKC-mediated phospho-rylation of Cx43, can reduce gap junctional intercellular communication activity in bovine aortic SMCs (Kuroki et al., 1998).
Diabetes and hyperglycemia may induce an increase in expression end secretion of ET-1 in vascular tissues as well (Kalani, 2008). Investigation of ET-1 in the development of abnormal retinal hemodynamics in DM showed that overexpression of ET-1 is associated with PKC activation, presumably PKC-ß2 and PKC-S isoforms (Park et al., 2000). It has been also reported that increase in contraction in response to ET-1 in the aorta and coronary arteries of rats is associated with augmented PKC activation in type 1 DM (Hattori et al., 1999; Tickerhoof et al., 2003).
Cyclooxygenases (COX)-derived prostanoids, which are arachidonic acid metabolites, also can play a significant role in diabetic vascular complications and have been implicated in hyperglycemia-induced endothelial dysfunction (Cosentino et al., 2003; Aljofan and Ding, 2010; Ishida et al., 2012). As has been shown in human aortic endothelial cells, hyperglycemia causes PKC-dependent increase in expression of inducible COX-2 isoform associated with an increase in TXA2 and a reduction of PGI2 release (Cosentino et al., 2003). In this mechanism glucose-induced activation of PKC may result in eNOS-dependent formation of peroxy-nitrite and tyrosine nitration and inactivation of PGI2 synthase (PGIS) (Cosentino et al., 2003). Production of vasoactive eicosanoid PGE2 can be also increased in diabetic vessels (Xia et al., 1996).
Conclusions
The present study shows that type 1 DM leads to increase in sensitivity to PhE in the pulmonary artery of rats reflecting enhancement in vascular apadrenoceptor-mediated contractility and that mechanisms of such enhancement in DM are associated with activity of PKC in the endothelium rather than in vascular SMCs. These mechanisms and the role of endothelium-mediated signaling in this process remain to be investigated in future studies.
References
Abebe, W., & McLeod, K. M. (1991). Enhanced arterial contractility to noradrenaline in diabetic rats is associated with increased phosphoinositide metabolism. Canadian Journal of Physiology and Pharmacology, 49, 355-361. Aljofan, M., & Ding, H. (2010). High glucose increases expression of cyclooxygenase-2, increases oxidative stress and decreases the generation of nitric oxide in mouse microvessel endothelial cells. Journal of Cellular Physiology, 222, 669-675. Beckman, J. A., Goldfine, A. B., Gordon, M. B., Garrett, L. A., & Creager, M. A. (2002). Inhibition of protein kinase C beta prevents impaired endothelium-dependent vasodilation caused by hyperglycemia in humans. Circulation Research, 90, 107-111. Bohlen, H. G., & Nase, G. P. (2001). Arteriolar nitric oxide concentration is decreased during hyperglycemia-induced betaII PKC activation. American Journal of Physiology. Heart and Circulatory Physiology, 280, H621-627.
Boussageon, R., Bejan-Angoulvant, T., Saadatian-Elahi, M., Lafont, S., Bergeonneau, C., Kassai, B., Erpeldinger, S., Wright, J. M., Gueyffier. F., & Cornu, C. (2011). Effect of intensive glucose lowering treatment on all cause mortality, cardiovascular death, and microvascular events in type 2 diabetes: Meta-analysis of randomised controlled trials. British
Medical Journal, 343, d4169.
Brisset, A. C., Isakson, B. E., & Kwak, B. R. (2009). Connexins in vascular physiology and pathology. Antioxidants and Redox Signaling, 11, 267-282.
Chang, K. S. K., & Stevens, W. C. (1992). Endothelium-dependent increase in vascular sensitivity to phenylephrine in long-term streptozotocin diabetic rat aorta. British Journal of Pharmacology, 107, 983-990.
Chen, W. G., Zhu, X. F., Hou, J. Q., Pu, J. X., & Yan, C. Y. (2008). Expressions of eNOS and connexin 43 in the penile tissue of rats with diabetic erectile dysfunction. Zhonghua Nan Ke Xue, 14, 427-430.
Chow, W. L., Zhang, L., & MacLeod, K. M. (2001). Noradrenaline-induced changes in intracellular Ca2+ and tension in mesenteric arteries from diabetic rats. British Journal of Pharmacology, 134, 179-187.
Clarke, M., & Dodson, P. M. (2007). PKC inhibition and diabetic microvascular complications. Best Practice and Research: Clinical Endocrinology and Metabolism, 21, 573-586.
Cosentino, F., Eto, M., De Paolis, P., van der Loo, B., Bachschmid, M., Ullrich, V., Kouroedov, A., Delli Gatti, C., Joch, H., Volpe, M., & Luscher, T. F. (2003). High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothelial cells: role of protein kinase C and reactive oxygen species. Circulation, 107, 1017-1023.
Cosentino-Gomes, D., Rocco-Machado, N., & Meyer-Fernandes, J. R. (2012). Cell signaling through protein kinase C oxidation and activation. International Journal of Molecular Sciences, 13, 10697-10721.
Cosson, E., Pham, I., Valensi, P., Paries, J., Attali, J. R., & Nitenberg, A. (2006). Impaired coronary endothelium-dependent vasodilation is associated with microalbuminuria in patients with type 2 diabetes and angiographically normal coronary arteries. Diabetes Care, 29, 107-112.
Feletou, M., & Vanhoutte, P. (2010). Endothelium-derived hyperpolarising factors and associated pathways: a synopsis. Pflugers Archiv -European Journal of Physiology, 459, 863-879.
Figueroa, X. F., & Duling, B. R. (2009). Gap junctions in the control of vascular function. Antioxidants and Redox Signaling, 11, 251-266.
Fleming, I., Fisslthaler, B., Dimmeler, S., Kemp, B. E., & Busse, R. (2001). Phosphorylation of Thr495 regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. Circulation Research, 88, E68-75.
Funk, S. D., Yurdagul, Jr. A.,& Orr, A W. (2012). Hyperglycemia and endothelial dysfunction in atherosclerosis: Lessons from type 1 diabetes. International Journal of Vascular Medicine, 2012, 1-19.
Gao, X., Martinez-Lemus, L. A., & Zhang, C. (2011). Endothelium-derived hyperpolarizing factor and diabetes. World Journal of Cardiology, 3, 25-31.
Georgescu, A., Alexandra, N., Constantinescu, E., & Popov, D. (2006). Effect of gap junction uncoupler heptanol on resistance arteries reactivity in experimental models of diabetes, hyperlipemia and hyperlipemia-diabetes. Vascular Pharmacology, 44, 513-518.
Geraldes, P., & King, G. L. (2010). Activation of protein kinase C isoforms and its impact on diabetic complications. Circulation Research, 106, 1319-1331.
Gurney, A. M., & Howarth, F. C. (2009). Effects of streptozotocin-induced diabetes on the pharmacology of rat conduit and resistance intrapulmonary arteries. Cardiovascular Diabetology, 8, 1-10.
Hattori, Y., Kawasaki, H., & Kanno, M. (1999). Increased contractile responses to endothelin-1 and U46619 via a protein kinase C-mediated nifedipine-sensitive pathway in diabetic rat aorta. Research Communications in Molecular Pathology and Pharmacology, 104, 73-80.
Herbert, M., Augereau, J. M., Gleye, J., & Maffrand, J. P. (1990). Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications, 172, 993-999.
Hink, U., Li, H., Mollnau, H., Oelze, M., Matheis, E., Hartmann, M., Skatchkov, M., Thaiss, F., Stahl, R. A., Warnholtz, A., Meinertz, T., Griendling, K., Harrison, D. G., Forstermann, U., & Munzel, T. (2001). Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circulation Research, 88, E14-22.
Hirata, K., Kuroda, R., Sakoda, T., Katayama, M., Inoue, N., Suematsu, M., Kawashima, S., & Yokoyama, M. (1995). Inhibition of endothelial nitric oxide synthase activity by protein kinase C. Hypertension, 25, 180-185.
Hou, C. J., Tsai, C. H., Su, C. H., Wu, Y. J., Chen, S. J., Chiu, J. J., Shiao, M. S., & Yeh, H. I. (2008). Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction. Journal of Histochemistry and Cytochemistry, 56, 745-752.
Inoguchi, T., Li, P., Umeda, F., Yu, H. Y., Kakimoto, M., Imamura, M., Aoki, T., Etoh, T., Hashimoto, T., Naruse, M., Sano, H., Utsumi, H., & Nawata, H. (2000). High glucose level and free fatty acid stimulate reactive oxygen
species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes, 49, 1939-1945.
Inoguchi, T., Ueda, F., Umeda, F., Yamashita, T., & Nawata, H. (1995). Inhibition of intercellular communication via gap junction in cultured aortic endothelial cells by elevated glucose and phorbol ester. Biochemical and Biophysical Research Communications, 208, 492-497.
Inoguchi, T., Yu, H. Y., Imamura, M., Kakimoto, M., Kuroki, T., Maruyama, T., & Nawata, H. (2001). Altered gap junction activity in cardiovascular tissues of diabetes. Medical Electron Microscopy, 34, 86-91.
Ishida, K., Matsumoto, T., Taguchi, K., Kamata, K., & Kobayashi, T. (2012). Protein kinase C delta contributes to increase in EP3 agonist-induced contraction in mesenteric arteries from type 2 diabetic Goto-Kakizaki rats. European Journal of Physiology, 463, 593-602.
Ishii, H., Jirousek, M. R., Koya, D., Takagi, C., Xia, P., Clermont, A., Bursell, S. E., Kern, T. S., Ballas, L. M., Heath, W. F., Stramm, L. E., Feener, E. P., & King, G. L. (1996). Amelioration of vascular dysfunctions in diabetic rats by an oral PKC p inhibitor. Science, 272, 728-731.
Kalani, M. (2008). The importance of endothelin-1 for microvascular dysfunction in diabetes. Vascular Health and Risk Management, 4, 1061-1068.
Kizub, I. V., Klymenko, K. I., & Soloviev, A. I. (2014). Protein kinase C in enhanced vascular tone in diabetes mellitus. International Journal of Cardiology, 174, 230-242.
Kizub, I. V., Pavlova, O. O., Johnson, C. D., Soloviev, A. I., & Zholos, A. V. (2010). Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats. British Journal of Pharmacology, 159, 1724-1731.
Klymenko, K. I., Novokhatska, T. V., Kizub, I. V., Parshikov, A. V., Dosenko, V.
E., & Soloviev, A. I. (2014). PKC-8 isozyme gene silencing restores vascular function in diabetic rat. Journal of Basic and Clinical Physiology and Pharmacology, 27, 1-9.
Kolluru, G. K., Bir, S. C., & Kevil, C. G. (2012). Endothelial dysfunction and diabetes: Effects on angiogenesis, vascular remodeling, and wound healing. International Journal of Vascular Medicine., 2012, 1-30.
Kuroki, T., Inoguchi, T., Umeda, F., Ueda, F., & Nawata, H. (1998). High glucose induces alteration of gap junction permeability and phosphorylation of connexin-43 in cultured aortic smooth muscle cells. Diabetes, 47, 931-963.
Lee, J.-H., Bahk, J.-H., Park, S.-H., & Huh, J. (2011). The diabetes-induced functional and distributional changes of the alpha 1-adrenoceptor of the abdominal aorta and distal mesenteric artery from streptozotocin-induced diabetic rats. Korean Journal of Anesthesiology, 60, 272-281.
Leo, C. H., Hart, J. L., & Woodman, O. L. (2011). Impairment of both nitric oxide mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocininduced diabetes. British Journal of Pharmacology, 162, 365-377.
Li, A. F., & Roy, S. (2009). High glucose-induced downregulation of connexin 43 expression promotes apoptosis in microvascular endothelial cells. Investigative Ophthalmology and Visual Science, 50, 1400-1407.
Lin, D., & Takemoto, D. J. (2005). Oxidative activation of protein kinase Cgamma through the C1 domain: Effects on gap junctions. Journal of Biological Chemistry, 280, 13682-13693.
Lopez-Lopez, J. G., Moral-Sanz, J., Frazziano, G., Gomez-Villalobos, M. J., Flores-Hernandez, J., Monjaraz, E., Cogolludo, A., & Perez-Vizcaino,
F. (2008). Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction. American journal of Physiology. Lung Cellular and Molecular, 295, L727-L732.
Madonna, R., & De Caterina, R. (2011). Cellular and molecular mechanisms of vascular injury in diabetes - part I: Pathways of vascular disease in diabetes. Vascular Pharmacology, 54, 68-74.
Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., & Dillmann, W. H.
(2008). Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice. American Journal of Physiology. Cell Physiology, 295, C221-C230.
Matsubara, M., Hayashi, N., Jing, T., & Titani, K. (2003). Regulation of endothelial nitric oxide synthase by protein kinase C. Journal of Biochemistry, 133, 773-781.
Matsumoto, T., Ishida, K., Nakayama, N., Kobayashi, T., & Kamata, K.
(2009). Involvement of NO and MEK/ERK pathway in enhancement of endothelin-1-induced mesenteric artery contraction in later-stage type 2 diabetic Goto-Kakizaki rat. American Journal of Physiology. Heart and Circulatory Physiology, 296, H1388-1397.
Matsumoto, T., Kobayashi, T., & Kamata, K. (2006). Mechanisms underlying the impaired EDHF type relaxation response in mesenteric
arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats. European Journal of Pharmacology, 538, 132-140.
Mehta, N. N., Sheetz, M., Price, K., Comiskey, L., Amrutia, S., Iqbal, N.. Mohler, E. R., & Reilly, M. P. (2009). Selective PKC beta inhibition with ruboxistaurin and endothelial function in type-2 diabetes mellitus. Cardiovascular Drugs and Therapy, 23, 17-24.
Michell, B. J., Chen, Z. P., Tiganis, T., Stapleton, D., Katsis, F., Power, D. A., Sim, A. T., & Kemp, B. E. (2001). Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein pinase. Journal of Biological Chemistry, 276, 1762517658.
Mueed, I., Zhang, L., & MacLeod, K. M. (2005). Role of the PKC/CPI-17 pathway in enhanced contractile responses of mesenteric arteries from diabetic rats to a-adrenoceptor stimulation. British Journal of Pharmacology, 146, 972-982.
Nobe, K., Sakai, Y., Nobe, H., & Momose, K. (2003). Dysfunction of aorta involves different patterns of intracellular signalingpathways in diabetic rats. European Journal of Pharmacology, 471, 195-204.
Oku, H., Kodama, T., Sakagami, K., & Puro, D. G. (2001). Diabetes-induced disruption of gap junction pathways within the retinal microvasculature. Investigative Ophthalmology and Visual Science, 42, 1915-1920.
Park, J. Y., Takahara, N., Gabriele, A., Chou, E., Naruse, K., Suzuma, K., Yamauchi, T., Ha, S. W., Meier, M., Rhodes, C. J., & King, G. L. (2000). Induction of endothelin-1 expression by glucose: An effect of protein kinase C activation. Diabetes, 49, 1239-1248.
Pitocco, D., Zaccardi, F., Di Stasio, E., Romitelli, F., Santini, S. A., Zuppi, C., & Ghirlanda, G. (2010). Oxidative stress, nitric oxide, and diabetes. Review of Diabetic Studies, 7, 15-25.
Ramana, K. V., Friedrich, B., Tammali, R., West, M. B., Bhatnagar, A., & Srivastava, S. K. (2005), Requirement of aldose reductase for the hyperglycemic activation of protein kinase C and formation of diacylglycerol in vascular smooth muscle cells. Diabetes, 54, 818-829.
Sato, T., Haimovici, R., Kao, R., Li, A. F., & Roy, S. (2002). Downregulation of connexin 43 expression by high glucose reduces gap junction activity in microvascular endothelial cells. Diabetes, 51, 1565-1571.
Somlyo, A. P., & Somlyo, A. V. (2003). Ca2+ sensitivity of smooth muscleand nonmuscle myosin II: Modulated by G proteins, kinases, and myosin phosphatase. Physiological Reviews, 83, 1325-1358.
Suzuma, K., Takahara, N., Suzuma, I., Isshiki, K., Ueki, K., Leitges, M., Aiello, L. P., & King, G. L. (2002). Characterization of protein kinase C beta isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization. Proceedings of the National Academy of Sciences of the United States of America, 99, 721-726.
Tickerhoof, M. M., Farrell, P. A., & Korzick, D. H. (2003). Alterations in rat coronary vasoreactivity and vascular protein kinase C isoforms in Type 1 diabetes. American Journal of Physiology. Heart and Circulatory Physiology, 285, H2694-703.
White, R. E., & Carrier, G. O. (1990). Vascular contraction induced by activation of membrane calcium ion channels is enhanced in streptozotocin-diabetes. Journal of Pharmacology and Experimental Therapeutics, 253, 1057-1062.
Whiteley, L., Padmanabhan, S., Hole, D., & Isles, C. (2005). Should diabetes be considered a coronary heart disease risk equivalent? Results from 25 years of follow-up in the Renfrew and Paisley survey. Diabetes Care, 28, 1588-1593.
Xavier, F. E., Davel, A. P. C., Rossoni, L. V., & Vassallo, D. V. (2003). Time-dependent hyperreactivity to phenylephrine in aorta from untreated diabetic rats: Role of prostanoids and calcium mobilization. Vascular Pharmacology, 40, 67-76.
Xia, P., Aiello, L. P., Ishii, H., Jiang, Z. Y., Park, D. J., Robinson, G. S., Takagi, H., Newsome, W. P., Jirousek, M. R., & King, G. L. (1996). Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. Journal of Clinical Investigation, 98, 2018-2026.
Yamada, Y., & Yokota, M. (1997). Effects of protein kinase C activation and inhibition on endothelin-1 release from human aortic and pulmonary artery endothelial cells comparison with effects on bovine endothelin-1 and human prostaglandin I2 release. American Journal of Hypertension, 10, 32-42.
Zimmet, P. Z. (2011). The growing pandemic of type 2 diabetes: A crucial need for prevention and improved detection. Medicographia, 33, 15-21.
