Zoltan Ungvari, Pal Pacher, Valeria Kecskemeti, Gyorgy Papp, Lajos Szollar, Akos Koller*

Transcription

1 Cardiovascular Research 43 (1999) locate/ cardiores locate/ cardiores Increased myogenic tone in skeletal muscle arterioles of diabetic rats. Possible role of increased activity of smooth muscle Ca channels and protein kinase C Zoltan Ungvari, Pal Pacher, Valeria Kecskemeti, Gyorgy Papp, Lajos Szollar, Akos Koller* From the Institutes of Pathophysiology, Pharmacology, and Department of Urology, Semmelweis University of Medicine, Budapest, Hungary and Department of Physiology, New York Medical College, Valhalla, New York, NY 10595, USA Abstract Received 14 October 1998; accepted 26 February 1999 Objective: The diabetes mellitus-induced microangiopathy is still not clearly characterized. In this study we aimed to elucidate the effect of streptozotocin (STZ)-induced diabetes on myogenic response of isolated rat skeletal muscle arterioles and the mechanisms responsible for its alterations. Methods: Male rats were divided into two groups: (1) control rats (C, plasma glucose: mmol/ l, n540) 2) diabetic rats (DM, 65 mg/kg STZ iv, plasma glucose: mmol/l, n540). Changes in diameter of isolated, cannulated gracilis skeletal muscle arterioles ( 130 mm in diameter) were measured by video-microscopy. Results: Step increases in perfusion pressure (PP; from 10 to 140 mmhg) elicited significantly greater constrictions in DM than in C gracilis arterioles, in the presence of the endothelium (E). Also, a step increase in PP (from 40 to 100 mmhg) elicited greater and faster constrictions in DM vs. C arterioles. There were no significant differences in the pressure-passive diameter (in Ca free solution) curves of arterioles. Dilations to acetylcholine were impaired in arterioles of DM as compared to those of C rats (EC 50, C: mol/l, DM: mol/l ( p,0.01), and unaffected by inhibition of nitric oxide synthesis with L-NNA (10 24 mol/l). Arteriolar constrictions to norepinephrine (NE) were significantly greater in DM compared to those of C rats (EC 50, C: mol/l, DM: mol/l, p,0.01) both in the presence and absence of E. In the absence of the E, constrictions to increases in pressure, or Ca ( mmol/l), or the 0 29 voltage-dependent Ca -channel agonist Bay K 8644 (EC 50; DM: mol/ l, C: mol/ l, p,0.05) or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA, EC 50; DM: mol/ l, C: mol/ l, p,0.05) were significantly greater in arterioles of DM compared to those of C rats. Conclusion: The novel findings of our study are that in diabetes mellitus the myogenic response of rat skeletal muscle arterioles is enhanced, which seems to be independent from the impaired endothelial function present simultaneously, and likely due to the increased activity of voltage-dependent Ca channels and/ or upregulation of protein kinase C in arteriolar smooth muscle Elsevier Science B.V. All rights reserved. Keywords: Microcirculation; Diabetes mellitus; Intraluminal pressure; Calcium sensitivity; Calcium channels; Protein kinase C; Rat Downloaded from at Pennsylvania State University on February, Introduction of hypertension, myocardial infarction and stroke [1 3]. Although the multifactorial effects of diabetes on the The microangiopathy described in diabetes mellitus is various regulatory mechanisms governing the diameter of still not well characterized. Studies in both human and blood vessels are not well understood, it is likely that experimental diabetes mellitus have demonstrated changes alterations in vascular reactivity precede the structural in blood vessel function which are thought to underlie changes seen in the established phase of diabetic miretinopathy, nephropathy, neuropathy and increase the risk croangiopathy. Alterations in the local regulation of skeletal muscle arteriolar tone [4 6] can lead to microvascular *Correspondence author. Tel.: ; fax: Time for primary review days / 99/ $ see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S (99)

2 Z. Ungvari et al. / Cardiovascular Research 43 (1999) disturbances contributing to the development of diabetic dependent Ca channels and protein kinase C (PKC) in foot syndrome described in diabetes mellitus [1 3]. smooth muscle known to be involved in the development One of the important mechanisms that regulates vascular of the myogenic response. resistance is the myogenic response which operates primarily in microvessels [7,8]. There are few studies, however, which have investigated the possible alteration of the myogenic response in diabetes, especially in skeletal 2. Methods muscle microcirculation. In vivo studies of cremasteric Diabetes was induced in male Wistar rats (n540, arterioles of streptozotocin (STZ)-induced diabetic rats weighing 120 g) by a single caudal intravenous injection demonstrated an impaired myogenic constrictor response of streptozotocin (STZ, 65 mg/kg) dissolved in 0.1 mol/l [9]. In isolated cremaster arterioles of rats, Hill and Ege citrate buffer. Non-diabetic control animals (C, n540) [10] showed that both passive and active characteristics of were injected with an equivalent volume of vehicle. All pressure-induced responses of arterioles have been rats were allowed to drink a 10% glucose solution in the changed in STZ-induced diabetes, and concluded that first 24 h after injection of STZ. Animals were housed myogenic response is impaired. separately, fed standard rat chow, and had free access to In certain conditions the function of the endothelium can drinking water. Blood glucose concentration was measured modify [8,11] the myogenic responses of skeletal muscle by glucose oxidase method once a week. Rats were arterioles. Previous studies have shown, that diabetes considered as diabetic when blood glucose levels were impairs endothelial functions resulting in reduced NO-.20 mmol/ l and glycosuria was present. All experiments mediated dilation to ACh [10,13 18] and constriction to were performed in accordance with the Guiding Principles increases in perfusate flow [12]. Furthermore, in the Care and Use of Animals published by the US Zimmermann et al. [19] have demonstrated that impaired National Institutes of Health (NIH Publication No , endothelial NO synthesis elicits an increased myogenic revised 1985). constriction of cerebral arteries isolated from STZ-induced diabetic female rats. The differences between the impaired [10] and enhanced [19] myogenic responses could be due 2.1. Isolation of arterioles to the fact that the characteristics of the myogenic response are different in various microvascular beds [7,8,19,20] and On the 12th week of diabetes, experiments were conthat diabetes-induced alterations in myogenic response is ducted on isolated first-order arterioles (approximately 130 organ specific due to the redistribution of blood flow in mm diameter) of rat gracilis muscle as previously described diabetes [4]. Furthermore recent studies indicate a greater [8]. Rats were anesthetized with intraperitoneal injection of role for endothelium in modifying the myogenic response sodium pentobarbital (50 mg/ kg) and the gracilis muscle of arterioles from female rats compared to that of males was exposed by an incision of the skin and isolated from [,22] which may also explain the different alterations of surrounding tissues. The muscle was then dissected out the myogenic response in the above-mentioned studies. and placed in a silicone-lined Petri dish containing cold There are several reports demonstrating alterations of (0 48C) physiological salt (PS) solution composed of Ca signaling in the smooth muscle of large vessels (mmol/ l): 110 NaCl, 5.0 KCl, 2.5 CaCl 2, 1.0 MgSO 4, 1.0 eliciting enhanced contractile response to agonists in KH2PO 4, 10.0 dextrose and 24.0 NaHCO3 and was diabetes [23 25]. Some of these pathways are known to be equilibrated with a gas mixture of 10% O2 and 5% CO 2, involved in the mechanotransduction of intravascular balanced with nitrogen, at ph 7.4. Using microsurgery pressure changes, yet the possible effect of these altera- instruments and an operating microscope, a segment 1.5 tions on the myogenic response of skeletal muscle ar- mm in length, of the first-order arteriole running interioles have not been clearly elucidated. On the basis of tramuscularly was isolated, and transferred in an organ these findings and our preliminary observations indicating chamber containing two glass micropipettes filled with PS an enhanced response of skeletal muscle arterioles to solution. From a reservoir the vessel chamber (15 ml) was increases in intraluminal pressure we hypothesized that continuously supplied with PS solution at a rate of 40 these alterations would rather enhance than impair the ml/ min. After the vessel had been mounted on the myogenic response of skeletal muscle arterioles in diabetes proximal micropipette and was secured with sutures, the mellitus. perfusion pressure was raised to 20 mmhg to clear the Thus, first we aimed to characterize the alterations of clotted blood from the lumen. Then the other end of the myogenic response in arterioles of gracilis skeletal muscle vessel was mounted on the distal pipette. Both micropipetof STZ-diabetic rats. The second aim of this study was to tes were connected with silicone tubing to an adjustable elucidate the mechanisms responsible for the alteration in reservoir. Pressure on both sides were measured by an myogenic responsiveness of these arterioles in diabetes, by electromanometer. The perfusion pressure was slowly assessing the synthesis/ release of endothelium-derived (approximately over 1 min) increased to 80 mmhg. The nitric oxide, and characterizing the function of voltage- temperature was set at 378C by a temperature controller Downloaded from at Pennsylvania State University on February, 2013

3 1020 Z. Ungvari et al. / Cardiovascular Research 43 (1999) (Grant Instruments, Great Britain) and the vessel was allowed to equilibrate for approximately 1 h. before and after the administration of the air bolus. The infusion of air resulted in loss of function of the endothelium, as indicated by the absence of dilation to ACh, whereas dilation to SNP remained intact Experimental protocols In other experiments a Ca -free PS solution was used to superfuse the vessels from control and diabetic rats until Only those vessels which developed spontaneous tone in maximal dilation developed, then changes in diameter in response to increases of perfusion pressure were used and 24 response to increasing concentrations of Ca (10 to thus no vasoactive agent was added to the PS solution to mol/ l) were assessed before and after removal establish arteriolar tone. After the equilibration period of the endothelium. In further experiments responses to changes in the diameter of arterioles in response to cumulative doses of the voltage-dependent Ca channel increases in perfusion pressure (from 10 to 140 mmhg, in 1 activator Bay K 8644 ( mol/ l) and the 10 mmhg steps) were measured under zero-flow con- protein kinase C (PKC) activator phorbol 12-myristate 0 27 ditions. The pressure was maintained for 5 10 min at each 13-acetate (PMA, mol/ l) were obtained pressure step to allow the vessel to reach a steady-state after removal of the endothelium. Diameter changes to diameter. At the conclusion of each experiment, the each dose of PMA were recorded for min to obtain suffusion solution was changed to a Ca - free PS maximal responses. 24 solution, which contained sodium nitroprusside (SNP, 10 Moreover, the vasoactive function of endothelium and mol/ l) and EGTA (ethylene glycol-bis(b-aminoethyl smooth muscle was assessed by peak responses of ar ether)-n,n,n,n -tetraacetic acid, 10 mol/ l). The vessel terioles to cumulative doses of ACh (10 to 10 mol/ l; 0 26 was incubated for 10 min, and the pressure steps were an endothelium-dependent agent), SNP (10 to 10 repeated to obtain the maximum passive diameter at each mol/ l; an endothelium-independent agent) and norepinephpressure value (pressure-passive diameter relationship). 0 rine (NE, from to 10 mol/ l). All drugs were Measurements of internal diameter at the midpoint of the added to the vessel chamber, and final concentrations are arteriolar segment were made using a high resolution CCD reported. After responses to each drug subsided the system video camera (Sony SSC M-370-CE) connected to a was flushed with PS solution. The vessel was than microscope (Carl Zeiss, Jena) and a video calliper caliv 24 incubated with N -nitro-l-arginine (L-NNA; 10 mol/ l) brated using a stage micrometer [8]. The diameter was for 30 min and responses to ACh and SNP were reassesrecorded with a chart recorder (Cole-Palmer). sed. In a further series of experiments, constrictor re- In other experiments, pressure was increased from 40 sponses of arterioles of control and diabetic rats to NE mmhg to 100 mmhg in one step, and the time course of were compared before and after removal of the endothe development of myogenic constriction was recorded thelium. for 5 min. Then the suffusion solution was changed to a All salts and chemicals were obtained from Sigma- 23 Ca - free PS solution, which contained EGTA (10 Aldrich and were prepared on the day of the experiment. mol/ l), the vessel was incubated for 10 min, and the Bay K 8644 was dissolved in ethanol and protected from pressure step (from 40 to 100 mmhg) was repeated. light; experiments were carried out in the dark. PMA was Diameters were normalized to the passive diameter at 100 dissolved in ethanol. The vehicle did not have vasoactive mmhg pressure. In order to characterize the dynamics of effect. the myogenic response a mono-exponential curve was fitted to the time-diameter data according to the following (2t /t) formula of Sipkema et al. [26] y 5 axe 1 d where a 2.3. Data analysis is the amplitude of the myogenic constriction, d isthe plateau diameter (as a percentage of the PD ) and t is Dilations were expressed as a percentage of the maximal 100 the time constant indicating the speed of the myogenic dilation of the vessel defined as the passive diameter at 80 constriction. mmhg perfusion pressure in Ca -free media containing In a second series of experiments the myogenic tone of 10 mol/ l EGTA and 10 mol/ l SNP. Constrictions arterioles from control and diabetic rats was compared in were expressed as a percentage of baseline. From the the absence of the endothelium. The endothelium of the cumulative dose response curves of vasoactive agents, the arteriole was removed by perfusion of the vessel with air half-maximal effective concentrations (EC 50) were calcufor 1 min at a perfusion pressure of 20 mmhg, as lated (sigmoidal non linear least-squares fit). Data are described earlier [8,11] The arteriole was then perfused expressed as means6sem, except for the regression with PS solution to clear the debris. The perfusion pressure parameters where SD values are given. Statistical analyses was then raised to 80 mmhg for 30 min to establish a were performed by two-way analysis of variance stable tone. The efficacy of endothelial denudation was (ANOVA) for repeated measures followed by Tukey s post ascertained by arteriolar responses to acetylcholine (ACh; hoc test or Student s t-test. P,0.05 was considered mol/ l), and sodium nitroprusside (SNP; 10 mol/ l) statistically significant. Downloaded from at Pennsylvania State University on February, 2013

4 Z. Ungvari et al. / Cardiovascular Research 43 (1999) Results 3.1. Effects of STZ-induced diabetes on standard parameters of rats Glycosuria developed within 24 h of STZ administration and was maintained throughout the 12-week period of diabetes. Within 1 wk of injection, fasting blood glucose levels exceeded 20 mmol/ l in over 95% of the rats receiving STZ. After 12 weeks all STZ treated rats had glycosuria and elevated plasma glucose levels as compared to controls (C: mmol/l; DM: mmol/l). Body weights of diabetic rats were depressed relative to controls (C: and DM: grams). There was no significant difference between the systolic blood pressure of control and STZ-diabetic rats (10463 and mmhg, respectively) Pressure diameter relationships of arterioles Isolated first-order arterioles of gracilis muscle from control rats developed active tone in response to step increases in PP ( mmhg) without the use of any vasoactive agent (Fig. 1, panel A). Initially, the diameter of these vessels increased from 100 mm to 150 mm in response to an increase in intravascular pressure from 10 to 40 mmhg. Beyond this point, further increases in pressure resulted in constriction of arterioles. Similarly, pressure diameter relationships were obtained in arterioles from Fig. 1. Panel A: Diameters of isolated skeletal muscle arterioles from diabetic animals. Initially, the diameter of diabetic arfilled control (C, n510, open symbols) and STZ-diabetic rats (DM, n510, symbols) as a function of perfusion pressure in the presence (circles) terioles increased similarly as controls. Further increases in and absence (triangles) of Ca in the superfusion solution. Panel B: pressure from 40 to 140 mmhg however, elicited a Changes in normalized diameter of arterioles as a function of perfusion significantly greater constriction of arterioles from diabetic pressure (C: open symbols, DM: filled symbols). Changes in diameter are rats (Fig. 1, panel A). In the absence of Ca (and in the normalized to the passive diameter at the corresponding pressure. Inset: 24 presence of SNP 10 mol/ l) the pressure passive diamarterioles The slopes of selected segments of the pressure diameter curves of eter relationship in each arteriole was also obtained (Fig 1, are significantly different. Data are mean6sem. * indicates panel A). Step increases in pressure elicited continuous significant ( p,0.05) differences from control. increases in the diameter of arterioles, reaching a plateau at 100 mmhg. The pressure-passive diameter relationship of We also investigated the pressure-induced changes in arterioles from each group did not differ significantly. diameter as a function of time. When the perfusion In order to indicate the strength of myogenic tone, the pressure was increased in one step from 40 to 100 mmhg, diameter of arterioles in active and passive conditions were both control and diabetic arteriolar diameters increased to compared (see Methods). Fig. 1, panel B depicts the the same extent, and then the diameters decreased gradualchanges in the diameter of arterioles as a percentage of ly for 3 5 min as depicted in Fig. 2 Panel A and B. The their passive diameter at corresponding perfusion original records indicate markedly greater pressure-induced pressures. We found that arterioles from diabetic rats constriction of the arteriole from the diabetic rat as exhibited a significantly decreased normalized diameter at compared to control. The summary data of the change in pressure values from 40 to 140 mmhg as compared to diameter in response to a pressure step in arterioles from controls. To further characterize the alteration of pressure- control and diabetic rats is shown in Fig. 2 Panel C. A induced tone in diabetes, the steepness of the descending mono-exponential curve was fitted to the time-diameter segment of the pressure diameter curves were also com- curves which showed a good fit for all responses. Compared. We found that the slope of this curve was sig- parison of the responses revealed that the amplitude a of nificantly greater in the arterioles from diabetic rats (Fig. the pressure-induced constriction was significantly greater 1, panel B, inset) as compared to controls (slopes: C: in arterioles from diabetic rats than those from control rats and DM: ; p,0.01; data are (C: % and DM: %, data are means6sd, means6sd). p,0.05). The plateau diameter d was significantly lower Downloaded from at Pennsylvania State University on February, 2013

5 1022 Z. Ungvari et al. / Cardiovascular Research 43 (1999) Fig. 2. Representative tracings of the changes in diameter of skeletal muscle arterioles from control (Panel A) and STZ-diabetic (Panel B) rats in response to a step increase in the perfusion pressure (PP) from 40 to 100 mmhg. Panel C: Changes in diameter of isolated skeletal muscle arterioles from control (C, n510, open symbols) and STZ-diabetic rats (DM, n510, filled symbols) as a function of time, in response to a step increase in pressure from 40 to 100 mmhg. Changes in diameter are normalized to the passive diameter obtained in Ca -free solution at 100 mmhg. Data are mean6sem. * Indicates significant ( p,0.05) differences from control. induced constriction is greater to a pressure step, but also the speed of its development is faster in arterioles of diabetic rats as compared to controls. To assess the possible modulatory role of endothelium in the enhanced constrictions to pressure increases, we removed the endothelium of arterioles. We found that infusion of air resulted in loss of the endothelium, as indicated by the absence of dilation to ACh, while dilation to SNP remained intact (arteriolar diameters at 80 mmhg after removal of the endothelium: C: mm, DM: mm). We found that removal of the endothelium did not significantly affect the pressure diameter curve of arterioles from either group of rats and did not change the difference between the pressure-induced constriction of arterioles from control and diabetic rats (Fig. 3, slopes in inset: C: ; DM: ; p,0.05; M6SD) Responses to ACh and SNP In a dose-dependent manner ACh (10 to 10 mol/l) elicited significantly greater dilations of arterioles isolated from control than those from diabetic rats (Fig. 4, panel A; EC x10 mol/ l and x10 mol/ l ( p,0.01), respectively). After preincubation (for 30 min) and in the presence of the nitric oxide synthase inhibitor 24 L-NNA (10 mol/ l) ACh-induced dilations of arterioles 28 from control (EC mol/ l; Fig. 4, panel 28 B), but not from diabetic rats (EC mol/ l; Fig. 4 panel C) decreased significantly. In a dosedependent manner SNP dilated arterioles of control and 29 diabetic rats (Fig. 4; EC mol/ l and 50 Downloaded from at Pennsylvania State University on February, 2013 in arterioles from diabetic than those from control rats (C: %, and DM: %, data are means6sd, Fig. 3. Changes in normalized diameter of isolated, endothelium-denuded p,0.05). The time constant t was significantly longer in skeletal muscle arterioles from control [C(2E), n56, open symbols] and STZ-diabetic rats [DM(2E), n56, filled symbols] as a function of arterioles from control than from diabetic rats (C: perfusion pressure. Inset: The slopes of selected segments of the , and DM:.263.1, M6SD, p,0.05). These pressure diameter curves of arterioles are significantly different. Data are data indicate that not only the maximum of the pressure- mean6sem. * Indicates significant ( p,0.05) differences from control.

6 Z. Ungvari et al. / Cardiovascular Research 43 (1999) Fig. 4. Panel A: Effect of cumulative doses of acetylcholine (ACh) on the normalized diameter of arterioles isolated from control (C, open symbols, v 24 n510) and STZ-diabetic rats (DM, filled symbols, n59). Panels B and C: Effect of N -nitro-l-arginine (L-NNA, 10 mol/ l, [(2E), triangles], a nitric oxide synthase inhibitor on ACh-induced responses in arterioles isolated from control and DM rats, respectively. Panel D: The effect of cumulative doses of sodium nitroprusside (SNP) on the normalized diameter of arterioles from control (C, open symbols, n510) and STZ-diabetic rats (DM, filled symbols, n59) rats in the presence and absence of L-NNA. Data are mean6sem. * Indicates significant ( p,0.05) differences from control mol/ l, respectively). L-NNA did not affect less, after removal of the endothelium, arterioles from dilations to SNP in either group (EC50 for C: diabetic rats were still significantly more responsive to mol/ l, DM: mol/ l, Fig. 4). norepinephrine than arterioles from control rats (Fig. 5, panel D) Responses to norepinephrine Downloaded from at Pennsylvania State University on February, In a dose-dependent manner norepinephrine (10 to 3.5. Responses to extracellular Ca mol/ l) elicited significantly greater constrictions in arterioles from diabetic than those from control rats Arterioles maximally dilated in a Ca -free PS solution (EC x10 mol/l and mol/l Administration of Ca (10 to mol/ l) con- ( p,0.01), respectively. (Fig. 5, panel A). Endothelium stricted arterioles from control and diabetic rats in a dose- removal enhanced constrictions of arterioles of control rats dependent manner. The dose response curve to Ca was 27 (EC mol/l, Fig. 5, panel B) whereas it characterized by a steep part indicating substantial con had no significant effect in arterioles from diabetic rats striction (from 10 to 10 mol/l) and a plateau phase (EC mol/ l, Fig. 5, panel C). Neverthe- (from 10 to mol/ l), where the arteriolar tone 50

7 1024 Z. Ungvari et al. / Cardiovascular Research 43 (1999) Fig. 5. Panel A: Effect of cumulative doses of norepinephrine (NE) on the normalized diameter of arterioles isolated from control (C, n510, open symbols) and STZ-diabetic rats (DM, n511, filled symbols). Panel B and C: Effect of endothelium removal (2E; triangles) on responses to NE in arterioles from C and DM rats. Panel D: Comparison of NE-induced responses of endothelium-denuded arterioles of C and DM rats. Data are mean6sem. * Indicates significant ( p,0.05) differences from control. was maintained. In arterioles of diabetic rats, Ca elicited PMA elicited significantly greater constrictions than those 29 significantly greater constrictions than those from controls from controls (EC mol/ l and 28 (Fig. 6, panel A). After removal of the endothelium, mol/ l ( p,0.05), respectively; Fig. 7, constrictions to Ca remained significantly increased in panel A). arterioles from diabetic rats compared to those from controls (Fig. 6, panel B). 4. Discussion Downloaded from at Pennsylvania State University on February, Responses to voltage-dependent Ca -channel The novel findings of our study are that (1) in skeletal opener and protein kinase C activator muscle arterioles of diabetic rats the pressure-sensitive myogenic response is enhanced, (2) this enhancement is In a dose-dependent manner the voltage-dependent Ca present in the absence of endothelium, (3) and most likely 1 channel activator Bay K 8644 (10 27 to 10 mol/ l) due to the increased activation of voltage-dependent Ca elicited significantly greater constrictions in endothelium- channels and/ or of protein kinase C in the arteriolar denuded arterioles from diabetic than those from control smooth muscle. Both the impairment of nitric oxide rats (EC mol/ l and mediation, as indicated by reduced dilations to acetylmol/ l ( p,0.05), respectively; Fig. 7, panel A). The PKC choline, and alteration of smooth muscle function are 0 27 activator phorbol 12-myristate 13-acetate ( likely to contribute to the enhanced constrictions of mol/ l) induced slow, concentration-dependent constric- skeletal muscle arterioles to norepinephrine in diabetes tions. In endothelium-denuded arterioles of diabetic rats mellitus.

8 Z. Ungvari et al. / Cardiovascular Research 43 (1999) Several mechanisms have been proposed to explain the microvascular disease observed in diabetes. Previous studies described how diabetes alters vessel wall morphol- Fig. 6. Panel A: Effect of increasing CaCl2 concentrations on the normalized diameter of arterioles from control (C, open symbols, n510) and STZ-diabetic rats (DM, filled symbols, n57). Panel B: Effect of increasing calcium concentrations on the normalized diameter of ar- terioles from control and DM rats after the removal of the endothelium (2E). Data are mean6sem. * Indicates significant ( p,0.05) differences from control. Fig. 7. Panel A: Effect of cumulative doses of Bay K 8644 on the normalized diameter of endothelium-denuded arterioles isolated from control (C, n55, open symbols) and STZ-diabetic rats (DM, n55, filled symbols). Panel B: Effect of cumulative doses of phorbol 12-myristate 13-acetate (PMA) on the normalized diameter of endothelium-denuded arterioles isolated from control (C, n55, open symbols) and STZ-diabetic rats (DM, n55, filled symbols). Data are mean6sem. * Indicates significant ( p,0.05) differences from control. the development of peripheral vascular disease in skeletal ogy (e.g. nonenzymatic glycosylation reactions), increases muscle observed frequently in diabetes mellitus [1 3]. platelet activity, and promotes low density lipoprotein Thus, in the present study we have utilized isolated gracilis modification [27]. However, many of the data describing muscle arterioles from STZ-diabetic and normal rats and the morphological [27,28] and functional [14,29 31] alter- examined the changes in the myogenic response and ations in diabetes were obtained in studies of large vessels. assessed the possible role of endothelium and smooth Less data are available to assess the possible alterations in muscle. regulation of vasomotor tone of microvessels in diabetes independently from neural and hormonal factors. One of 4.1. Effect of diabetes on pressure-induced constrictions the primary regulators of arteriolar tone is the pressuresensitive myogenic response [7,8,20]. Because the skeletal In the present study we found that arterioles from muscle microcirculation represents a major part of diabetic rats exhibited enhanced constrictions to increases peripheral resistance and determine tissue blood flow, the in intraluminal pressure compared to that of controls as dysfunction of myogenic mechanisms may contribute to indicated by a steeper active pressure diameter relation- Downloaded from at Pennsylvania State University on February, 2013

9 1026 Z. Ungvari et al. / Cardiovascular Research 43 (1999) ship (more negative slope) and a higher maintained tone in arterioles isolated from skeletal muscle of diabetic rats, (Fig. 1). Zimmerman et al. [19] also reported an enhanced an impairment which is likely to be due to the reduced vascular tone during increases in perfusion pressure in biological availability of NO as suggested by previous isolated rat cerebral arteries of STZ-diabetic female rats. studies [12,13]. Dilatations in response to the NO donor Contrary to our findings in mesenteric arteries [12] and SNP were similar in vessels from control and diabetic rats. cremaster arterioles [10] an impaired myogenic response On the basis of the above mentioned studies and was reported. findings in small cerebral arteries of diabetic rats showing It is thought that one of the roles of myogenic mecha- that the pressure-induced tone is enhanced due to the lack nism is to prevent sudden increases in perfusion pressure of NO [19] one can assume that the impaired function of in the distal part of microcirculation by increasing arterio- endothelium may contribute to the enhanced myogenic lar resistance when pressure increases. Thus, we investi- constriction of gracilis arterioles in diabetes. However, in gated the time-dependent behavior of the myogenic re- the present study we found that removal of the endosponse as well. A step increase in perfusion pressure from thelium did not significantly affect the pressure-induced 40 to 100 mmhg resulted in an increase in diameter responses of gracilis arterioles either from control or followed by a constriction (Fig. 2). The pressure step was diabetic rats (Fig. 3). Earlier reports demonstrated little accomplished in less than two seconds, a time much faster modulatory role of endothelium on the myogenic tone of than the time constant of the myogenic response prevent- gracilis muscle arterioles of male rats under no-flow ing the myogenic mechanism to counteract the passive conditions [8]. This may explain why the lack of NO increase in diameter [26]. We found that in arterioles from release in endothelium-denuded vessels has no significant diabetic rats the time constant was significantly shorter and effect on the pressure diameter curve of skeletal muscle the amplitude of the response was significantly greater than arterioles from diabetic rats. The reason for the greater in arterioles from control rats (Fig. 2). The faster and modulatory role of NO on the basal tone of cerebral greater constrictions to increases in intravascular pressure vessels than on arterioles of skeletal muscle may be due to may exaggerate myogenic response in vivo during changes the fact that the cerebral vessels were isolated from female in blood pressure which may result in temporal reductions rats [19] whereas in our study we used vessels from male of blood supply to the skeletal muscle in diabetes [4]. rats. Indeed, previous studies showed a gender difference, Since myogenic mechanism is determined by passive i.e. greater release and effect of NO on myogenic tone of and active properties of the arteriolar wall, it can be rat in female arterioles compared to those of males [,22]. hypothesized that changes in myogenic response observed Thus, diabetes may affect the female and male carin the present study are due to alterations in the structure diovascular system differently as it was also shown in of vessels. In our study the pressure diameter curve of hypertension [22]. As the increase in pressure-induced tone arterioles from control and diabetic rats in Ca free PS in diabetic arterioles could not be related to the altered solution were not significantly different (Fig. 1), sug- function of endothelium, we hypothesized that the engesting no change in the passive compliance of arterioles hancement is due to factors intrinsic to arteriolar smooth which may modify active responses. Interestingly, Hill and muscle. Ege [10] showed changes in the passive mechanical properties of cremasteric arterioles and hypothesized that 4.2. Effect of diabetes on arteriolar responses to this could be due to advanced glycosylation or other norepinephrine changes in the structure of the vessel wall and contribute to the impaired myogenic response. However, it is likely that Arteriolar smooth muscle contractility can be characteralterations of cremasteric arterioles are different in diabetes ized by responses of arterioles to norepinephrine, an agent than those of skeletal muscle or other tissues [19] known to elicit receptor-mediated influx of extracellular Endothelial factors, such as NO, are known to im- Ca [25]. The findings that diabetes enhances arteriolar portantly modulate vascular tone. Impaired endothelium- constrictions to NE (Fig. 5, panel A) is in accordance with dependent, flow-mediated vasodilation and impaired vas- earlier data reporting increased responses to NE in aorta orelaxation to endothelium-dependent agents (such as [27] and mesenteric arteries [32,34,35] of STZ-induced acetylcholine) have been described both in diabetic patients diabetic rats. Present (Fig. 5, panel B) and previous studies [14,15] and in animal models of diabetes [10,13,16,18,19]. in skeletal muscle arterioles demonstrated that there is A reduced release of NO from endothelium in diabetes has concomitant release of endothelial factors upon administrabeen proposed at least in part to be responsible for the tion of NE [36]. NE-induced responses of arterioles from increased responses of large vessels and mesenteric mi- diabetic rats, however, remained unchanged after removal crovessels to constrictor agents [27,32,33]. Furthermore, of the endothelium (Fig. 5, panel C), suggesting an the increased myogenic tone of cerebral arteries from impaired endothelial function. Our finding that in diabetes STZ-diabetic rats has been recently attributed also to such constrictions to NE are significantly greater even in the impairment of endothelial function [19]. In the present absence of the endothelium (Fig. 5, panel D) extend study, we found that ACh-mediated vasodilation is reduced previous reports on mesenteric arteries from STZ-diabetic Downloaded from at Pennsylvania State University on February, 2013

10 Z. Ungvari et al. / Cardiovascular Research 43 (1999) rats [23,37] to skeletal muscle arterioles, and suggest that ly, (3) and it is likely due to the increased activity of the enhanced responsiveness of microvessels to a-adren- voltage-dependent Ca channels and/ or protein kinase C ergic agonists cannot be attributed only to dysfunction of in arteriolar smooth muscle. These alterations could lead to endothelium but also due to the altered Ca -signaling an increase in arteriolar tone in vivo eliciting reduced mechanisms in the smooth muscle [25,35]. microvascular perfusion of skeletal muscle, which may be of importance in the development of skeletal muscle 4.3. Altered calcium sensitivity and myogenic response microangiopathy associated with diabetes mellitus. in diabetes Earlier findings demonstrated that the maximum con- tractile response to Ca increases in renal [24] and Acknowledgements mesenteric [38,35] arteries of diabetic rats. Similarly, We thank Miriam Nunez and Mary Browne for excellent increased responses to Bay K 8644 in renal [24] and secretarial assistance. This study was supported by grants mesenteric [35,39] arteries of diabetic rats have been from the Hungarian National Science Research Foundation reported. It was also shown that both resting and NE- OTKA T , OTKA T , the Health Science 45 induced Ca uptake increases in renal arteries of Council ETT 524/ 97 and the National Heart, Lung, and diabetic rats [24]. Furthermore, there are studies suggesting Blood Institute USA HL that in diabetes mellitus the enhanced contraction of smooth muscle to various agonists may be attributed to the enhanced activation of protein kinase C [40 42]. Previous studies established that pressure-induced References myogenic constriction of skeletal muscle arterioles, at least [1] Kannel WB, McGee DL. Diabetes and cardiovascular disease: the in part, depends on the influx of extracellular Ca through Framingham Study. J Am Med Assoc 1979;241: voltage dependent Ca channels [43,44] and activation of [2] Garcia MJ, McNamara PM, Gordon T, Kannel WB. Diabetes as a PKC [20]. Thus, we compared the constrictions of gracilis cardiovascular risk factor. Diabetes 1974;23: arterioles to increasing concentrations of extracellular Ca [3] Ruderman NB, Gupta S, Sussman I. An overview. In: Ruderman and to pharmacological activation of voltage-gated Ca NB, Williamson JR, Brownlee ML, editors, Hyperglycemia, diabetes, and vascular disease, New York, NY: Oxford Press, 1992, pp. channels and PKC in the presence of myogenic tone developed in response to an intravascular pressure of 80 [4] Hill MA, Larkins RG. Alterations in distribution of cardiac output in mmhg. We found that constrictions to increasing con- experimental diabetes. Am J Physiol 1989;257:H571 H580. centrations of extracellular Ca were significantly greater [5] Hill MA, Meininger GA, Granger HJ. Altered skeletal muscle in arterioles from diabetic rats than in those of control rats, hemodynamics after one week of streptozotocin induced diabetes. a difference which was independent of the endothelium Microcirc Endothelium Lymphatics 1985;2: [6] Tooke JE. Microvascular function in human diabetes. Diabetes (Fig. 6). Similarly, constrictions to pharmacological stimu- 1995;44: lation of voltage-gated Ca channels by Bay K 8644 (in [7] Johnson PC. The myogenic response. In: Handbook of physiology. the absence of the endothelium) were also enhanced in The cardiovascular system. Vascular smooth muscle, Vol. II, arterioles from diabetic rats compared to those of controls Bethesda, MD: Am. Physiol. Soc, 1980, pp , sect 2, chap. (Fig. 7 panel A), suggesting that the enhanced activity of 15. [8] Sun D, Kaley G, Koller A. Characteristics and origin of myogenic these channels contribute to the enhanced myogenic re- response in isolated gracilis muscle arterioles. Am J Physiol sponse in diabetes. 1994;266:H1177 H1183. The mechanism by which PKC activation increases [9] Hill MA, Meininger GA. Impaired arteriolar myogenic reactivity in vascular tone is still unclear; it may involve sensitization early experimental diabetes. Diabetes 1993;42: of contractile proteins to Ca, increase in [Ca ] and [10] Hill MA, Ege EA. Active and passive mechanical properties of i isolated arterioles from STZ-induced diabetic rats. Effect of aminoincrease of myosin light chain phosphorylation [40] mech- guanidine treatment. Diabetes 1994;432: anisms that are important in the development of myogenic [11] Huang A, Sun D, Koller A. Endothelial dysfunction augments response [43,45 47]. Because in the present study arterio- myogenic arteriolar constriction in hypertension. Hypertension lar constrictions to PMA, a known activator of PKC, were 1993;22: enhanced in arterioles from diabetic rats (Fig. 7 panel B), [12] Tribe RM, Thomas CR, Poston L. Flow-induced dilation in isolated resistance arteries from control and streptozotocin-diabetic rats. the increased activity of PKC may also be responsible for Diabetologia 1998;41: the development of enhanced myogenic response in dia- [13] Alsip NL, Schuschke DA, Miller FN. Microvascular responses in betes mellitus. the skeletal muscle of the diabetic rat. J Lab Clin Med In summary, the novel findings of our study are that (1) 1996;128: experimental diabetes mellitus results in enhanced pres- [14] De Tejada IS, Goldstein I, Azadzoi K, Krane RJ, Cohen RA. Impaired neurogenic and endothelium-mediated relaxation of penile sure-induced myogenic constrictions in rat skeletal muscle smooth muscle from diabetic men with impotence. N Eng J Med arterioles, (2) this enhancement seems to be independent 1989;320: of the impaired endothelial function present simultaneous- [15] McNally PG, Watt PAC, Rimmer T et al. 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