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Am J Physiol Heart Circ Physiol 287: H1064-H1071, 2004. First published May 6, 2004; doi:10.1152/ajpheart.00069.2004
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Diabetes-related changes in cAMP-dependent protein kinase activity and decrease in relaxation response in rat mesenteric artery

Takayuki Matsumoto, Kentaro Wakabayashi, Tsuneo Kobayashi, and Katsuo Kamata

Department of Physiology and Morphology, Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan

Submitted 17 February 2004 ; accepted in final form 26 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Using superior mesenteric artery rings isolated from age-matched controls and streptozotocin (STZ)-induced diabetic rats, we recently demonstrated that EDHF-type relaxation is impaired in STZ-induced diabetic rats, possibly due to a reduced action of cAMP via increased phosphodiesterase (PDE) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283–H291, 2003). Here, we investigated the activity and expression of cAMP-dependent protein kinase (PKA), an enzyme that is produced by a pleiotropic and plays key roles in the transduction of many external signals through the cAMP second messenger pathway and in cAMP-mediated vasorelaxation. The relaxation induced by cilostamide, a selective PDE3 inhibitor, was significantly weaker in superior mesenteric artery rings from STZ-induced diabetic rats than in those from age-matched controls. The relaxation responses to 8-bromo-cAMP (8Br-cAMP) and N6,O2-dibutyryl-adenosine-cAMP (db-cAMP), a cell-permeant cAMP analog, were also impaired in the STZ diabetic group. PKA activity in the db-cAMP-treated mesenteric artery was significantly lower in the STZ diabetic group. The expression levels of the mRNA and protein for PKA catalytic subunit Cat-{alpha} were significantly decreased in the STZ diabetic group, but those for PKA regulatory subunit isoform RII-{beta} were increased. We conclude that the abnormal vascular relaxation responsiveness seen in STZ-induced diabetic rats may be attributable not only to increased PDE activity but also to decreased PKA activity. Possibly, the decreased PKA activity may result from an inbalance between PKA catalytic and regulatory subunit expressions.

protein kinase A; streptozotocin

ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE (cAMP) acts as a second messenger in the intracellular signal transduction of a wide variety of extracellular stimuli in several tissues (3, 39). In the vascular system, cAMP plays important roles in the regulation of vascular tone and in the maintenance of the mature contractile phenotype in smooth muscle cells (31, 41). The intracellular levels of cAMP are tightly regulated, both by control of its rate of synthesis (by adenylyl cyclases) in response to extracellular signals and by rate of control of its hydrolysis [by cyclic nucleotide phosphodiesterases (PDEs)] (3, 38, 39). The newly minted cAMP then binds to its receptor, cAMP-dependent protein kinase (PKA), and causes reversible phosphorylation of protein substrates that regulate a vast number of cellular processes, such as metabolism, cell growth and differentiation, apoptosis, gene expression, ion channel conductivity, and vascular tone (41, 53, 55).

There are two types of PKA, type I (PKA-I) and type II (PKA-II), and these share a common catalytic (C) subunit but contain distinct regulatory (R) subunits (RI and RII, respectively) (53, 55, 57). Through biochemical studies and gene cloning, four isoforms of the R subunits (RI-{alpha}, RI-{beta}, RII-{alpha}, and RII-{beta}) have been identified. Three distinct C subunits (Cat-{alpha}, Cat-{beta}, and Cat-{gamma}) have also been identified. In the holoenzyme state, PKA exists in an inactive form. After an increase in intracellular cAMP, the R subunits bind to cAMP, resulting in the dissociation of the holoenzyme into a regulatory dimer and two C monomers. The free C subunits can then phosphorylate various substrates. Thus both C and R subunits are important for PKA-mediated functions (30, 53). In addition to their biochemical differences, the isoforms of PKA may have different effects on a number of physiological and pathophysiological processes and signaling mechanisms in cells, and these may underlie the differential effects of cAMP. This is supported by, for instance, the finding that, although the total R subunits-to-C subunits ratio in normal tissue is relatively constant at around 1:1, the relative amounts of RI and RII vary and depend to a large extent on both physiological conditions and the hormonal status of the tissue (11, 19, 35, 54). One recent study (9) showed that in RII-{beta} gene knockout mice, an increased level of RI-{alpha} compensates for the loss of RII-{beta} in brown fat cells. The switching of the PKA isoform from type II to type I results in an increased basal level of PKA activity and increased energy expenditure. Such RII-{beta} knockout mice have been noted to be leaner and also protected against diet-induced obesity, insulin resistance, and dyslipidemia (2, 9, 51). These results demonstrate clearly that RI and RII are functionally distinct.

Diabetes mellitus is associated with vascular complications, including an impairment of vascular responsiveness to neurotransmitters in the macro- and microvasculature (10). In the macrovasculature, an accumulating body of evidence suggests that the relaxation responses induced in aortic strips by endothelium-dependent agents are weaker in streptozotocin (STZ)-induced diabetic rats than in nondiabetic control rats (8, 23, 26, 28, 45, 47, 49). Moreover, several reports have indicated that diabetic rats show an impairment of ACh-induced, endothelium-dependent hyperpolarization/relaxation in the microvasculature (16, 36, 62). The contribution made by EDHF to relaxation is dependent on vessel size, being more prominent in the smaller, physiologically more important arteries than in larger ones (5, 40). Thus an impairment of EDHF-mediated responses may have important implications for the mechanisms by which diabetes leads to vascular dysfunction because small vessel dysfunction (such as in retinopathy, nephropathy, and neuropathy) is one of the major complications seen in diabetes. Recently, we demonstrated that EDHF-type relaxation was impaired in STZ-induced diabetic rats and that this impairment might be attributable to a reduction in the action of cAMP via increased PDE activity (37). There are several reports indicating that cAMP-mediated responses, such as {beta}-receptor-mediated responses, are altered in diabetic states. For example, Type I diabetic patients showed a decreased {beta}-adrenergic responsiveness of the heart beat in isoproterenol infusion experiments (4). In ventricular cardiomyocytes and papillary muscle from STZ diabetic rats, the {beta}-adrenergic stimulatory pathway involves an additional defect upstream of the adenylate cyclase/G protein system (25, 61). Furthermore, in the vascular system, the isoproterenol-induced relaxation response is impaired in the STZ diabetic aorta (24). However, no study has yet investigated the relationship between diabetic vasculopathy and the cAMP downstream (cAMP-PKA) system.

In the present study, we designed experiments to investigate the diabetes-related changes in cAMP-induced vasodilation in the rat superior mesenteric artery. We were especially interested in PKA activity in diabetes. We also asked whether mesenteric arteries from control and established diabetic rats might differ in their PKA subunit expression profiles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. STZ, phenylephrine, indomethacin, NG-nitro-L-arginine (L-NNA), 3-isobutyl-1-methylxanthine (IBMX), 8-bromo-cAMP (8Br-cAMP), N6,O2-dibutyryl-cAMP (db-cAMP), EDTA, EGTA, PMSF, leupeptin, aprotinin, monoclonal {beta}-actin antibody, and protease inhibitor cocktail were all purchased from Sigma Chemical (St. Louis, MO). Cilostamide was from Calbiochem-Novabiochem (La Jolla, CA). All drugs were dissolved in saline except where otherwise noted (e.g., IBMX and cilostamide were dissolved in DMSO). Horseradish peroxidase (HRP)-linked secondary anti-mouse and anti-rabbit antibodies were purchased from Promega (Madison, WI), whereas antibodies for PKA catalytic and regulatory subunit isoforms (Cat-{alpha}, RI, RII-{alpha}, and RII-{beta}) were from BD Biosciences (San Jose, CA). PKA Cat-{beta} subunit antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Animals and experimental design. Male Wistar rats (8 wk old and 180–230 g body wt) received a single injection via the tail vein of 65 mg/kg STZ dissolved in a citrate buffer. Age-matched control rats were injected with the buffer alone. Food and water were given ad libitum. This study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University (which is accredited by the Ministry of Education, Science, Sports and Culture, Japan).

Measurement of plasma glucose. Twelve weeks after the injection of STZ or buffer, plasma glucose was determined using a commercially available enzyme kit, which made use of the O-toluidine method (Wako Chemical; Osaka, Japan).

Measurement of isometric force. Vascular isometric force was recorded as in our previous paper (37). Rats were anesthetized with diethyl ether and euthanized by decapitation 12 wk after treatment with STZ or buffer. The superior mesenteric artery was rapidly removed and immersed in oxygenated, modified Krebs-Henseleit solution (KHS). This solution consisted of (in mM) 118.0 NaCl, 4.7 KCl, 25.0 NaHCO3, 1.8 CaCl2, 1.2 NaH2PO4, 1.2 MgSO4, and 11.0 dextrose. The artery was carefully cleaned of all fat and connective tissue, and ring segments 2 mm in length were suspended by a pair of stainless steel pins in a well-oxygenated (95% O2-5% CO2) bath of 10 ml KHS at 37°C. The rings were stretched until an optimal resting tension of 1.0 g was loaded and then allowed to equilibrate for at least 60 min. Force generation was monitored by means of an isometric transducer (model TB-611T, Nihon Kohden; Tokyo, Japan). The tissues were equilibrated for 40 min in the presence of 100 µM L-NNA and 10 µM indomethacin (to block nitric oxide synthase and cyclooxygenase, respectively) before the administration of phenylephrine (1 µM). Once the phenylephrine-induced contraction had stabilized, vasodilator responses were elicited either in a cumulative manner (cilostamide, a specific inhibitor of PDE3) or in a single concentration-effect manner [8Br-cAMP (a cell-permeable cAMP analog rated as having greater resistance to hydrolysis by PDE) and db-cAMP (a cell-permeable cAMP analog), the latter in the presence of 10 µM IBMX, a nonselective inhibitor of PDE]. In the experiments with IBMX, an equieffective concentration of phenylephrine was used (1–10 µM).

In vitro kinase assay for PKA activity. Mesenteric arteries were pretreated with 20 µM IBMX for 30 min at 37°C and then treated with 100 µM db-cAMP or vehicle (deionized water) for 30 min at 37°C. They were then rapidly frozen in liquid N2 and stored at –80°C. The mesenteric tissues were homogenized in a homogenization buffer containing (in mM) 25 Tris·HCl (pH 7.4), 150 NaCl, 1 EDTA, 1 EGTA, 0.3 PMSF, 0.04 leupeptin, and 0.02 aprotinin. The homogenates were centrifuged, and the supernatant (10 µg protein) was used in a nonradioactive assay for PKA (PKA assay system, Promega). For the positive control (10 ng PKA; provided with the kit), we used the same assay conditions as for the artery samples. For the negative control (deionized water), we again used the same assay conditions as for the artery samples. Phosphorylated and nonphosphorylated peptide bands were visualized on a 0.8% agarose gel, and the former were quantitated by spectrophotometry.

Enzyme immunoassay for cAMP. Mesenteric rings from diabetic and age-matched control rats were incubated for 30 min at 37°C in oxygenated KHS containing 20 µM IBMX and then incubated for 30 min at 37°C with either 100 µM db-cAMP or vehicle. The stimulated level of cAMP was then determined. To this end, rings were rapidly frozen in liquid N2 and stored at –80°C. cAMP was then extracted in 6% trichloroacetic acid, followed by neutralization with water-saturated diethyl ether, and an enzyme immunoassay (Amersham Biosciences UK; Little Chalfont, UK) was performed.

Measurement of expressions of mRNAs for PKA subunit isoforms using RT-PCR. RNA was isolated by the guanidinium method (7). Briefly, rat superior mesenteric arteries were carefully isolated and then cleaned of fat and connective tissue. The arteries were homogenized in RNA buffer, and the RNA was quantified by ultraviolet absorbance spectrophotometry. For the RT-PCR analysis, first-strand cDNA was synthesized from total RNA using oligo(dT)20 and a ThermoScript RT-PCR System (Invitrogen; Carlsbad, CA). All primers were synthesized by Sigma-Genosys (St. Louis, MO). Individual sequences, PCR conditions, product size, and GenBank accession numbers are shown in Table 1. The PCR products so obtained were analyzed on ethidium bromide-stained agarose (1.5%) gels. The PCR products were quantified by scanning densitometry, with the amount of each product being normalized with respect to the amount of GAPDH product.


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  		Table 1. Oligonucleotide primer sequences for PKA subunit isoforms and GAPDH and PCR protocols

 
Western blot analysis. Mesenteric arterial tissues (three pooled vessels) were homogenized in ice-cold homogenization buffer containing 20 mM Tris·HCl (pH 7.5), 1 mM MgCl2, and protease inhibitor cocktail. Homogenates were centrifuged at 3,000 g for 5 min. The supernatant was collected, and the proteins were solubilized in Laemmli's buffer containing mercaptoethanol. The protein concentration was determined by means of a bicinchoninic acid (BCA) protein assay reagent kit (Pierce). Samples (20 µg/lane) were resolved by electrophoresis on 12% SDS-PAGE gels and then transferred onto polyvinylidene difluoride membranes. Briefly, after the residual protein sites on the membrane were blocked with Block ace (Dainippon-pharm; Osaka, Japan), the membrane was incubated with anti-PKA Cat-{alpha} (1:1,000), anti-PKA Cat-{beta} (1:500), anti-PKA RI (1:250), anti-PKA RII-{alpha} (1:1,000), or anti-PKA RII-{beta} (1:1,000) in blocking solution. HRP-conjugated anti-mouse or anti-rabbit antibody was used at a 1:10,000 dilution in Tween-PBS, followed by detection using SuperSignal (Pierce). To normalize the data, we used {beta}-actin as a housekeeping protein. The {beta}-actin protein levels were determined after the membrane was stripped and probed with {beta}-actin monoclonal primary antibody (1:5,000), with HRP-conjugated anti-mouse IgG as the secondary antibody. The optical densities of the bands on the film were quantified using densitometry, with correction for the optical density of the corresponding {beta}-actin band.

Statistical analysis. Data are expressed as means ± SE. When appropriate, statistical differences were assessed by Dunnett's test for multiple comparisons after one-way ANOVA, with a probability level of P < 0.05 being regarded as significant. Statistical comparisons between concentration-response curves were made using a two-way ANOVA, with Bonferroni's correction for multiple comparisons being performed post hoc (P < 0.05 again being considered significant).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Blood glucose levels and animal body weights. As reported previously (37), at the time of the experiment, all STZ-treated rats exhibited hyperglycemia, their blood glucose concentrations (599.1 ± 26.4 mg/dl, n = 12) being significantly higher than those of the age-matched nondiabetic control rats [99.8 ± 6.4 mg/dl, n = 12 (P < 0.001)]. The body weights of the diabetic rats (240.2 ± 4.3 g, n = 12) were significantly lower than those of the age-matched control rats [518.9 ± 21.7 g, n = 12 (P < 0.001)] at the time of the experiment.

PDE inhibitor-induced relaxation. cAMP signaling in mammalian cells is terminated by cyclic nucleotide PDEs, a multifamily class of enzymes that catalyze the hydrolysis of cyclic nucleotides to 5'-nucleotide monophosphates (which do not activate cAMP effector proteins; Refs. 3 and 38). Among the various PDEs, the two of greatest interest are PDE3 and PDE4 because they preferentially hydrolyze cAMP (48). To investigate cAMP-mediated relaxation in the rat mesenteric artery, we first tested the effect of cilostamide (10–9–10–5 M), a specific PDE3 inhibitor, when it was added cumulatively to rings precontracted by phenylephrine (1 µM) in the presence of 100 µM L-NNA plus 10 µM indomethacin. The tension developed in response to 1 µM phenylephrine was 2.16 ± 0.15 g in diabetic mesenteric rings (n = 7) and 2.34 ± 0.06 g in those from the age-matched controls (n = 7, no significant difference). The concentration-response curves (Fig. 1) showed that the peak relaxation induced by cilostamide was significantly weaker in mesenteric arteries from diabetic rats [63.9 ± 6.9% and 84.6 ± 3.3% of the phenylephrine-induced tone in diabetic (n = 7) and age-matched controls (n = 7), respectively (P < 0.05)]. The EC50 values for the cilostamide-induced relaxations were 232.7 ± 26.9 and 141.6 ± 10.1 nM in diabetic and age-matched controls, respectively (P < 0.01). On the other hand, when Ro 20-1724 (10–9–10–5 M), a PDE4-specific inhibitor, was added cumulatively to rings precontracted by phenylephrine (1 µM) in the presence of 100 µM L-NNA plus 10 µM indomethacin, it induced almost no relaxation in either of the two (data not shown).



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  		Fig. 1. Concentration-response curves for the relaxations induced by cilostamide (phosphodiesterase 3-specific inhibitor) in isolated rings of superior mesenteric artery obtained from age-matched controls and diabetic rats. Ordinate shows relaxation of the superior mesenteric artery as a percentage of the contraction induced by phenylephrine (1 µM). In each experiment, NG-nitro-L-arginine (L-NNA; 100 µM) + indomethacin (10 µM) was applied 40 min before the phenylephrine application and was present thereafter. Each data point represents the mean ± SE from 7 experiments; the SE is included only when it exceeds the dimension of the symbol used. *P < 0.05, diabetic vs. control.

 
Effects of cAMP analogs on relaxation responses. To clarify the cAMP-mediated vasodilation, we examined the relaxing effect of a cAMP analog on rings from diabetic and age-matched control rats. In the presence of 100 µM L-NNA plus 10 µM indomethacin, 8Br-cAMP (1 mM), a cell-permeant cAMP analog that is rated as more resistant to PDEs, caused a relaxation of mesenteric artery rings from age-matched controls, the peak being by 23.4 ± 2.2% (n = 8). However, the relaxation response to this dose of 8Br-cAMP was significantly smaller in the diabetic group [11.7 ± 1.8%, n = 6 (P < 0.01); Fig. 2A]. We also examined the effect of another cAMP analog (db-cAMP) in the presence of IBMX, a PDE inhibitor. As IBMX depressed contraction, the concentration of phenylephrine used in experiments involving IBMX was increased to between 1 and 10 µM. The tension developed in response to 1–10 µM phenylephrine in the presence of IBMX was 1.90 ± 0.09 g in diabetic mesenteric rings (n = 7) and 1.95 ± 0.09 g in the age-matched control group (n = 7, no significant difference). The peak amplitude of the relaxation induced by 30 µM db-cAMP in rings pretreated with IBMX was significantly smaller in the diabetic group than in the age-matched control group [79.6 ± 4.96% and 92.7 ± 1.73% of the phenylephrine-induced tone in diabetic (n = 7) and age-matched control (n = 7) groups, respectively (P < 0.05); Fig. 2B].



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  		Fig. 2. Decrease in cAMP derivative-induced relaxation in diabetic mesenteric arteries. In each experiment, L-NNA (100 µM) plus indomethacin (10 µM) was applied 40 min before the phenylephrine application and was present thereafter. A: 8-bromo-cAMP (8Br-cAMP; 1 mM) was added after the 1 µM phenylephrine-induced contraction had reached a steady-state level. B: N6,O2-dibutyryl-cAMP (db-cAMP; 30 µM) was added after the induction of contraction by 1–10 µM phenylephrine in the presence of 10 µM IBMX. Results are expressed as means ± SE; n = 6–8. *P < 0.05 and **P < 0.01, diabetic vs. control.

 
Effects of cAMP analogs on PKA activity and cAMP accumulation in rat mesenteric artery. PKA has been shown to contribute to cAMP signaling and to its impact on vascular function (41). As reported previously (37), PDE activity is increased in the diabetic mesenteric artery. We therefore examined the effects of db-cAMP treatment on PKA activity, after pretreatment with the PDE inhibitor IBMX (20 µM), in mesenteric artery rings. As shown in Fig. 3, there was no significant difference in vehicle-treated PKA activity between the diabetic and age-matched control groups. After stimulation with 100 µM db-cAMP, PKA activity was significantly increased in the age-matched control group [1.08 ± 0.25 and 1.96 ± 0.17 U/ml in vehicle- and db-cAMP-treated mesenteric arteries, respectively (P < 0.05)] but not in the diabetic group (1.24 ± 0.14 and 1.31 ± 0.23 U/ml in vehicle- and db-cAMP-treated mesenteric arteries, respectively). The db-cAMP-stimulated level of PKA activity in mesenteric arteries was significantly lower in the diabetic group than in the age-matched controls (P < 0.05).



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  		Fig. 3. Measurement of PKA activity in superior mesenteric arteries from control and diabetic rats. The mesenteric arteries were pretreated with 20 µM IBMX for 30 min, and, subsequently, 100 µM db-cAMP or vehicle (Veh) was added for 30 min. Details are given in MATERIALS AND METHODS. A: PKA assay was performed using PepTag assay protein kinase kits according to the manufacturer's instructions. The phosphorylation state of the peptide was examined by agarose gel electrophoresis. B: phosphorylated peptide bands were quantitated by spectrophotometry. Results are means ± SE from 6 experiments. *P < 0.05, db-cAMP-treated control vs. corresponding Veh; #P < 0.05, db-cAMP-treated control vs. db-cAMP-treated diabetic.

 
Next, to confirm that PKA received the same degree of stimulation in mesenteric arteries from age-matched controls and STZ-induced diabetic rats, cAMP levels were measured in rat mesenteric arterial rings that had first been treated with 20 µM IBMX for 30 min and then incubated for 30 min with 100 µM db-cAMP or vehicle (the conditions also used for PKA activity measurements). Under our conditions, vehicle-treated cAMP levels were not significantly different between diabetic and age-matched control groups [41.6 ± 7.4 and 47.7 ± 8.0 pmol/mg protein in diabetic (n = 3) and age-matched control (n = 3) rats, respectively]. Moreover, db-cAMP-treated cAMP levels were not significantly different between the two groups [92.0 ± 17.4 and 94.3 ± 10.7 pmol/mg protein in diabetic (n = 4) and age-matched control (n = 4) rats, respectively]. Thus our PKA activity assay system was successful at demonstrating similarities between the diabetic and control groups.

Expression of mRNAs and proteins for PKA subunits. To investigate the possible mechanisms underlying the impaired cAMP analog-induced PKA activity seen in mesenteric arteries from STZ-induced diabetic rats, we examined whether the expressions of mRNAs and proteins for the various PKA subunits (see Introduction) might be altered in the diabetic state. Using RT-PCR analysis on the total RNA isolated from superior mesenteric arteries from age-matched controls and STZ-induced diabetic rats, we found the following. The expression of GAPDH mRNA showed no difference between the diabetic and age-matched control groups, but the expression of mRNA for the PKA C subunit Cat-{alpha} was significantly lower in the diabetic rats (compared with the age-matched controls). On the other hand, the expression of mRNA for the PKA R subunit RII-{beta} was significantly increased in the diabetic group (Fig. 4). The expressions of mRNAs for the PKA Cat-{beta}, RI-{alpha}, and RII-{alpha} subunits showed no differences between the diabetic and age-matched control groups (Fig. 4).



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  		Fig. 4. RT-PCR assay of mRNA expressions levels for PKA subunit isoforms in superior mesenteric arteries from control and diabetic rats. A: expressions of mRNAs for PKA Cat-{alpha}, Cat-{beta}, RI-{alpha}, RII-{alpha}, and RII-{beta} subunits in control and diabetic rat mesenteric arteries, as assayed by RT-PCR. B: quantitative analysis of expressions of mRNAs for PKA subunits (by scanning densitometry). n = 5 control rats and 5 steptozotocin-induced diabetic rats. Values are means ± SE of 5 determinations (PKA subunits/GAPDH). **P < 0.01, diabetic vs. control. The RT-PCR assay was performed as described in MATERIALS AND METHODS. Each total RNA preparation (3.0 µg) was reverse transcribed, and the cDNA product was PCR amplified using various primers, with several cycles being employed. A portion of the PCR reaction product was electrophoresed on a 1.5% agarose gel containing ethidium bromide.

 
Immunoblot analysis of mesenteric arteries from diabetic and age-matched control rats (using anti-PKA subunit antibodies) allowed detection of immunoreactive proteins (Fig. 5). In parallel with the mRNA expression profiles, the PKA C subunit Cat-{alpha} protein levels were significantly lower in the diabetic group than in the age-matched controls, whereas the expression of the protein for the PKA R subunit RII-{beta} was significantly higher in the former than in the latter group. The expressions of the proteins for the PKA Cat-{beta}, RI-{alpha}, and RII-{alpha} subunits showed no differences between the diabetic and age-matched control groups (Fig. 5).



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  		Fig. 5. Analysis of protein expressions for PKA catalytic and regulatory subunit isoforms in superior mesenteric arteries from control and diabetic rats. A: representative Western blots of PKA subunit isoforms. Protein samples (20 µg) were subjected to 12% PAGE and then transferred to polyvinylidene difluoride membranes. These were then incubated with primary antibodies specific for each catalytic (Cat-{alpha}, 40 kDa; Cat-{beta}, 42 kDa) or regulatory (RI, 48 kDa; RII{alpha}, 51 kDa; RII-{beta}, 53 kDa) subunit isoform of PKA and with secondary anti-mouse or anti-rabbit antibodies. The membranes were stripped and probed with {beta}-actin (42 kDa) primary and secondary anti-mouse antibodies. B: bands were quantified as described in MATERIALS AND METHODS. Ratios were calculated for the optical densities of PKA subunit isoforms over that of {beta}-actin. Values are means ± SE of 3–6 determinations (subunits/{beta}-actin). *P < 0.05, diabetic vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we made two major findings related to 1) the impairment of cAMP-mediated relaxation and PKA activity in the mesenteric artery in STZ-induced diabetic rats, and 2) the abnormal pattern of expression of PKA subunits in that diabetic artery.

To assess cAMP-dependent relaxation in mesenteric arteries obtained from diabetic and age-matched control rats, we used cilostamide, which inhibits PDE3 and increases intracellular cAMP, 8Br-cAMP, and db-cAMP. Because complex interactions between different vasodilator pathways in vascular smooth muscle have been proposed for nitric oxide synthase and cyclooxygenase (32), we investigated vasodilator responses in the presence of inhibitors of these enzymes (L-NNA and indomethacin). In mammalian cells, cAMP signaling is terminated by PDEs (3, 38, 48). Of the PDE families identified in vascular smooth muscle cells, members of the PDE3 (cGMP inhibited) and PDE4 (cAMP specific) families have been shown to contribute to the regulation of cAMP signaling and its impact on vascular smooth muscle cell function (3, 38, 48). More specifically, inhibitors of PDE3 or PDE4 increase vascular smooth muscle cell cAMP levels, and an inhibition of PDE3 activity has marked effects on vascular smooth muscle cell contraction-relaxation coupling (i.e., it induces vasodilation). Several studies have found PDE3 activities to be altered in diabetes (42, 43), and our recent study (37) demonstrated that PDE3 activity in the mesenteric artery was increased in STZ-induced diabetic rats. In the present study, our first finding was that inhibition of PDE3 by cilostamide induced a relaxation response in the mesenteric artery that was attenuated in STZ-induced diabetic rats (Fig. 1). The concentration-response curve for cilostamide showed not only a significant rightward shift but also a significantly decreased maximum response in the diabetic group. Thus our data suggest that the impairment of cilostamide-induced relaxation seen in diabetic rats was not entirely attributable to increased PDE activity. On the other hand, an inhibition of PDE4 activity induced almost no vasodilation, as reported by others (34, 48). We speculated that the impairment of vascular responsiveness in the diabetic group might be attributable to an abnormality in the cAMP signaling pathway as well as to an increased PDE activity. To clarify this point, we studied cAMP analog-mediated relaxation. The first analog we tested, 8Br-cAMP, is effective at activating PKA but much less effective at activating PKG, and it is rated as being more resistant to PDE (15, 18). In our study, the 8Br-cAMP-mediated relaxation was significantly impaired in the diabetic group (Fig. 2). Furthermore, when we tested a second analog, we found the db-cAMP-mediated relaxation seen in the presence of the PDE inhibitor IBMX to also be reduced in the diabetic group (Fig. 2). Taken together, the above results suggest that in the diabetic mesenteric artery, relaxation is impaired downstream of cAMP.

PKA represents a key component in the cAMP signaling pathway, because it is responsible for the intracellular transduction of most cAMP-mediated physiological effects in a variety of cells (53, 55, 57). Activation of PKA in vascular smooth muscle cells by an elevated cytoplasmic cAMP concentration results in relaxation as a result of phosphorylations both of effector proteins, including myosin light chain kinase, and of the ion channels that regulate the cytosolic Ca2+ concentration and hyperpolarization (e.g., K+ channels) (41, 52, 58). Furthermore, PKA-mediated phosphorylations underpin the EDHF phenomenon in several arteries (21, 56). In the present study, cAMP analog-stimulated PKA activity was decreased in the diabetic mesenteric artery (Fig. 3), and, under the same conditions as those used for the PKA activity assay, cAMP levels in mesenteric arteries were very similar between control and diabetic rats. Therefore, we directly demonstrated that PKA activity was impaired in the diabetic mesenteric artery. This is consistent with the previous finding that PKA activity is altered in the diabetic state (12, 44). As mentioned above, it has been assumed that activation of PKA may represent one of the mechanisms by which cAMP and cAMP analogs induce vasorelaxation (17). On the other hand, several studies have suggested that PKA is not the sole mediator of cAMP-dependent vasodilation (13, 18, 59, 60). Although the precise mechanism by which cAMP causes relaxation in the rat mesenteric artery is not certain, our data indicate that the impairment of cAMP-mediated relaxation seen in the diabetic state may be, at least in part, due to a relative lack of PKA activity. Future experiments will need to address this question.

In the native state, PKA exists as a tetramer holoenzyme consisting of two R subunits and two C subunits. In the holoenzyme state, PKA is in an inactive form. After an increase in intracellular cAMP, the regulatory subunits bind to cAMP, which results in the dissociation of the holoenzyme into a R dimer and two C subunit monomers. The free catalytic subunits can then phosphorylate various substrates (30, 53, 55, 57). Here, we report, for the first time, decreased Cat-{alpha} protein and increased RII-{beta} protein in the diabetic mesenteric artery (as compared with controls) (Fig. 5). Similar changes in expression were seen at the mRNA level (Fig. 4), indicating transcriptional alterations in the diabetic state. These changes imply decreased PKA holoenzyme (R2C2) targeting and therefore decreased local pools of C subunits. These expression abnormalities may contribute to the decreased cAMP-induced PKA activity and vasodilation/relaxation observed in the diabetic mesenteric artery. This conclusion is supported by the findings showing that 1) the inactive holoenzyme can form at saturating cAMP levels, leading to decreased cAMP-responsive transcription, in cells overexpressing R subunits, and 2) maximally cAMP-stimulated cells overexpressing RI-{alpha} show less nuclear translocation of a microinjected C subunit (29, 30). Further support for this hypothesis is provided by the previous observations that in hepatocytes and neuroblastoma cells, cAMP signaling is decreased both by induction of the RI-{alpha} subunit and by degradation of the C subunit (20, 50).

Although the mechanism underlying such differences in the expressions and activities of PKA subunits between diabetic and control rats remains unclear, metabolic and/or hormonal alterations might be involved. In rats fed a low-protein diet, insulin secretion by the islets was decreased, and this impairment was associated with reduced PKA Cat-{alpha} subunit expression (14). Moreover, the expression of the gene that encodes the regulatory subunit of PKA was increased in this model (14). Furthermore, PKA RII-{beta} subunit knockout mice have been noted both to be leaner and to be protected against diet-induced obesity, insulin resistance, and dyslipidemia (2, 9, 51). These results suggest that alleles at loci coding for PKA regulatory subunits should be included in the list of candidate genes determining susceptibility both to obesity and to the diabetes associated with obesity. In our STZ diabetic model, long-term insulin deficiency and hyperglycemia are associated with metabolic abnormalities such as increases in plasma triglyceride, cholesterol, and low-density lipoproteins (27). In addition, the levels of counterregulatory hormones such as catecholamines, adrenocorticotropic hormone, and glucagon are markedly higher in the diabetic condition (1, 6). Thus it is possible that the observed abnormalities in the expression levels of PKA subunits may be due to changes in the plasma levels of the above hormones. This speculation is supported by the finding that PKA subunit expression is regulated by several hormones (22, 33, 46). However, to establish causal relationships will require research focusing, for example, on time-course changes both in the expressions of the mRNAs for PKA subunits and in PKA activity in diabetic state.

In conclusion, we found that cAMP-mediated relaxation was impaired in the STZ diabetic mesenteric artery and that this impairment may be attributable to reduced PKA activity, in turn, resulting from an alteration in the pattern of expression of PKA subunits. Future research into vascular signaling mechanisms should continue to focus on physiological and therapeutic means of stimulating the cAMP signal transduction cascade in diabetic vasculopathy.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported in part by the Ministry of Education, Science, Sports and Culture, Japan, and by the Promotion and Mutual Aid Cooperation for Private Schools in Japan, and by the Takeda Science Foundation.


   ACKNOWLEDGMENTS
 
The authors thank A. Iwasaki, T. Katohno, and N. Hirata for technical help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Kamata, Dept. of Physiology and Morphology, Institute of Medicinal Chemistry, Hoshi Univ., Shinagawa-ku, Tokyo 142-8501, Japan (E-mail: kamata{at}hoshi.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Adeghate E, Ponery AS, and Sheen R. Streptozotocin-induced diabetes mellitus is associated with increased pancreatic tissue levels of noradrenaline and adrenaline in the rat. Pancreas 22: 311–316, 2001.[CrossRef][ISI][Medline]
  2. Amieux PS, Cummings DE, Motamed K, Brandon EP, Wailes LA, Le K, Idzerda RL, and McKnight GS. Compensatory regulation of RI{alpha} protein levels in protein kinase A mutant mice. J Biol Chem 272: 3993–3998, 1997.[Abstract/Free Full Text]
  3. Beavo JA. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 75: 725–748, 1995.[Abstract/Free Full Text]
  4. Berlin I, Grimaldi A, Bosquet F, and Puech AJ. Decreased beta-adrenergic sensitivity in insulin-dependent diabetic subjects. J Clin Endocrinol Metab 63: 262–265, 1986.[Abstract]
  5. Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, and Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci 23: 374–380, 2002.[CrossRef][Medline]
  6. Chan O, Chan S, Inouye K, Shum K, Matthews SG, and Vranic M. Diabetes impairs hypothalamo-pituitary-adrenal (HPA) responses to hypoglycemia, and insulin treatment normalizes HPA but not epinephrine responses. Diabetes 51: 1681–1689, 2002.[Abstract/Free Full Text]
  7. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]
  8. Cohen RA. The role of nitric oxide and other endothelium-derived vasoactive substances in vascular disease. Prog Cardiovasc Dis 38: 105–128, 1995.[CrossRef][ISI][Medline]
  9. Cummings DE, Brandon EP, Planas JV, Motamed K, Idzerda RL, and McKnight GS. Genetically lean mice result from targeted disruption of the RII beta subunit of protein kinase A. Nature 382: 622–626, 1996.[CrossRef][Medline]
  10. De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, and Vanhoutte PM. Endothelial dysfunction in diabetes. Br J Pharmacol 130: 963–974, 2000.[CrossRef][ISI][Medline]
  11. Døskeland SO, Maronde E, and Gjertsen BT. The genetic subtypes of cAMP-dependent protein kinase–functionally different or redundant? Biochim Biophys Acta 1178: 249–258, 1993.[Medline]
  12. Dutta K, Carmody MW, Cala SE, and Davidoff AJ. Depressed PKA activity contributes to impaired SERCA function and is linked to the pathogenesis of glucose-induced cardiomyopathy. J Mol Cell Cardiol 34: 985–996, 2002.[CrossRef][ISI][Medline]
  13. Eckly-Michel A, Martin V, and Lugnier C. Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation. Br J Pharmacol 122: 158–164, 1997.[CrossRef][ISI][Medline]
  14. Ferreira F, Barbosa HC, Stoppiglia LF, Delghingaro-Augusto V, Pereira EA, Boschero AC, and Carneiro EM. Decreased insulin secretion in islets from rats fed a low protein diet is associated with a reduced PKAalpha expression. J Nutr 134: 63–67, 2004.[Abstract/Free Full Text]
  15. Francis SH, Noblett BD, Todd BW, Wells JN, and Corbin JD. Relaxation of vascular and tracheal smooth muscle by cyclic nucleotide analogs that preferentially activate purified cGMP-dependent protein kinase. Mol Pharmacol 34: 506–517, 1988.[Abstract]
  16. Fukao M, Hattori Y, Kanno M, Sakuma I, and Kitabatake A. Alterations in endothelium-dependent hyperpolarization and relaxation in mesenteric arteries from streptozotocin-induced diabetic rats. Br J Pharmacol 121: 1383–1391, 1997.[CrossRef][ISI][Medline]
  17. Haynes J Jr, Robinson J, Saunders L, Taylor AE, and Strada SJ. Role of cAMP-dependent protein kinase in cAMP-mediated vasodilation. Am J Physiol Heart Circ Physiol 262: H511–H516, 1992.[Abstract/Free Full Text]
  18. Hei YJ, MacDonell KL, McNeill JH, and Diamond J. Lack of correlation between activation of cyclic AMP-dependent protein kinase and inhibition of contraction of rat vas deferens by cyclic AMP analogs. Mol Pharmacol 39: 233–238, 1991.[Abstract]
  19. Hofmann F, Bechtel PJ, and Krebs EG. Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues. J Biol Chem 252: 1441–1447, 1977.[Abstract/Free Full Text]
  20. Houge G, Vintermyr OK, and Døskeland SO. The expression of cAMP-dependent protein kinase subunits in primary rat hepatocyte cultures. Cyclic AMP down-regulates its own effector system by decreasing the amount of catalytic subunit and increasing the mRNA for the inhibitory (R) subunits of cAMP-dependent protein kinase. Mol Endocrinol 4: 481–488, 1990.[Abstract]
  21. Imig JD, Inscho EW, Deichmann PC, Reddy KM, and Falck JR. Afferent arteriolar vasodilation to the sulfonimide analog of 11,12-epoxyeicosatrienoic acid involves protein kinase A. Hypertension 33: 408–413, 1999.[Abstract/Free Full Text]
  22. Jahnsen T, Lohmann SM, Walter U, Hedin L, and Richards JS. Purification and characterization of hormone-regulated isoforms of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovaries. J Biol Chem 260: 15980–15987, 1985.[Abstract/Free Full Text]
  23. Kamata K, Miyata N, and Kasuya Y. Impairment of endothelium-dependent relaxation and changes in levels of cyclic GMP in aorta from streptozotocin-induced diabetic rats. Br J Pharmacol 97: 614–618, 1989.[ISI][Medline]
  24. Kamata K, Miyata N, and Kasuya Y. Involvement of endothelial cells in relaxation and contraction responses of the aorta to isoproterenol in native and streptozotocin-induced diabetic rats. J Pharmacol Exp Ther 249: 890–894, 1989.[Abstract/Free Full Text]
  25. Kamata K, Satoh T, Tanaka H, and Shigenobu K. Changes in electrophysiological and mechanical responses of the rat papillary muscle to {alpha}- and {beta}-agonist in streptozotocin-induced diabetes. Can J Physiol Pharmacol 75: 781–788, 1997.[CrossRef][ISI][Medline]
  26. Kanie N, Matsumoto T, Kobayashi T, and Kamata K. Relationship between peroxisome proliferator-activated receptors (PPARalpha and PPARgamma) and endothelium-dependent relaxation in streptozotocin-induced diabetic rats. Br J Pharmacol 140: 23–32, 2003.[CrossRef][ISI][Medline]
  27. Kobayashi T and Kamata K. Relationship among cholesterol, superoxide anion and endothelium-dependent relaxation in diabetic rats. Eur J Pharmacol 367: 213–222, 1999.[CrossRef][ISI][Medline]
  28. Kobayashi T, Matsumoto T, and Kamata K. Mechanisms underlying the chronic pravastatin treatment-induced improvement in the impaired endothelium-dependent aortic relaxation seen in streptozotocin-induced diabetic rats. Br J Pharmacol 131: 231–238, 2000.[CrossRef][ISI][Medline]
  29. Kopperud R, Christensen AE, Kjarland E, Viste K, Kleivdal H, and Døskeland SO. Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions. J Biol Chem 277: 13443–13448, 2002.[Abstract/Free Full Text]
  30. Kopperud R, Krakstad C, Selheim F, and Døskeland SO. cAMP effector mechanisms. Novel twists for an ‘old’ signaling system. FEBS Lett 546: 121–126, 2003.[CrossRef][ISI][Medline]
  31. Koyama H, Bornfeldt KE, Fukumoto S, and Nishizawa Y. Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation. J Cell Physiol 186: 1–10, 2001.[CrossRef][ISI][Medline]
  32. Lamping K. Interactions between NO and cAMP in the regulation of vascular tone. Arterioscler Thromb Vasc Biol 21: 729–730, 2001.[Free Full Text]
  33. Levy FO, Ree AH, Eikvar L, Govindan MV, Jahnsen T, and Hansson V. Glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells. Endocrinology 124: 430–436, 1989.[Abstract]
  34. Lindgren SH, Andersson TL, Vinge E, Andersson KE. Effects of isozyme-selective phosphodiesterase inhibitors on rat aorta and human platelets: smooth muscle tone, platelet aggregation and cAMP levels. Acta Physiol Scand 140: 209–219, 1990.[ISI][Medline]
  35. Lohmann SM and Walter U. Regulation of the cellular and subcellular concentrations and distribution of cyclic nucleotide-dependent protein kinases. Adv Cyclic Nucleotide Protein Phosphorylation Res 18: 63–117, 1984.[ISI][Medline]
  36. Makino A, Ohuchi K, and Kamata K. Mechanisms underlying the attenuation of endothelium-dependent vasodilatation in the mesenteric arterial bed of the streptozotocin-induced diabetic rat. Br J Pharmacol 130: 549–556, 2000.[CrossRef][ISI][Medline]
  37. Matsumoto T, Kobayashi T, and Kamata K. Alterations in EDHF-type relaxation and phosphodiesterase activity in mesenteric arteries from diabetic rats. Am J Physiol Heart Circ Physiol 285: H283–H291, 2003.[Abstract/Free Full Text]
  38. Matsumoto T, Kobayashi T, and Kamata K. Phosphodiesterases in the vascular system. J Smooth Muscle Res 39: 67–86, 2003.[CrossRef][Medline]
  39. Maurice DH, Palmer D, Tilley DG, Dunkerley HA, Netherton SJ, Raymond DR, Elbatarny HS, and Jimmo SL. Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol Pharmacol 64: 533–546, 2003.[Abstract/Free Full Text]
  40. McGuire JJ, Ding H, and Triggle CR. Endothelium-derived relaxing factors: a focus on endothelium-derived hyperpolarizing factor(s). Can J Physiol Pharmacol 79: 443–470, 2001.[CrossRef][ISI][Medline]
  41. Murray KJ. Cyclic AMP and mechanisms of vasodilation. Pharmacol Ther 47: 329–345, 1990.[CrossRef][ISI][Medline]
  42. Nagaoka T, Shirakawa T, Balon TW, Russell JC, and Fujita-Yamaguchi Y. Cyclic nucleotide phosphodiesterase 3 expression in vivo: evidence for tissue-specific expression of phosphodiesterase 3A or 3B mRNA and activity in the aorta and adipose tissue of atherosclerosis-prone insulin-resistant rats. Diabetes 47: 1135–1144, 1998.[Abstract]
  43. Netherton SJ, Jimmo SL, Palmer D, Tilley DG, Dunkerley HA, Raymond DR, Russell JC, Absher PM, Sage EH, Vernon RB, and Maurice DH. Altered phosphodiesterase 3-mediated cAMP hydrolysis contributes to a hypermotile phenotype in obese JCR:LA-cp rat aortic vascular smooth muscle cells: implications for diabetes-associated cardiovascular disease. Diabetes 51: 1194–1200, 2002.[Abstract/Free Full Text]
  44. Netticadan T, Temsah RM, Kent A, Elimban V, and Dhalla NS. Depressed levels of Ca2+-cycling proteins may underlie sarcoplasmic reticulum dysfunction in the diabetic heart. Diabetes 50: 2133–2138, 2001.[Abstract/Free Full Text]
  45. Oyama Y, Kawasaki H, Hattori Y, and Kanno M. Attenuation of endothelium-dependent relaxation in aorta from diabetic rats. Eur J Pharmacol 132: 75–78, 1986.[CrossRef][ISI][Medline]
  46. Oyen O, Sandberg M, Eskild W, Levy FO, Knutsen G, Beebe S, Hansson V, and Jahnsen T. Differential regulation of messenger ribonucleic acids for specific subunits of cyclic adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase by cAMP in rat Sertoli cells. Endocrinology 122: 2658–2666, 1988.[Abstract]
  47. Pieper GM. Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension 31: 1047–1060, 1998.[Free Full Text]
  48. Polson JB and Strada SJ. Cyclic nucleotide phosphodiesterases and vascular smooth muscle. Annu Rev Pharmacol Toxicol 36: 403–427, 1996.[CrossRef][ISI][Medline]
  49. Poston L and Taylor PD. Endothelium-mediated vascular function in insulin-dependent diabetes mellitus. Clin Sci (Lond) 88: 245–255, 1995.[Medline]
  50. Prashad N, Lotan D, and Lotan R. Differential effects of dibutyryl cyclic adenosine monophosphate and retinoic acid on the growth, differentiation, and cyclic adenosine monophosphate-binding protein of murine neuroblastoma cells. Cancer Res 47: 2417–2424, 1987.[Abstract/Free Full Text]
  51. Schreyer SA, Cummings DE, McKnight GS, and LeBoeuf RC. Mutation of the RII{beta} subunit of protein kinase A prevents diet-induced insulin resistance and dyslipidemia in mice. Diabetes 50: 2555–2562, 2001.[Abstract/Free Full Text]
  52. Schubert R and Nelson MT. Protein kinases: turners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 22: 505–512, 2001.[CrossRef][Medline]
  53. Skalhegg BS and Tasken K. Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci 5: d678–d693, 2000.[CrossRef][Medline]
  54. Sugden PH and Corbin JD. Adenosine 3',5'-cyclic monophosphate-binding proteins in bovine and rat tissues. Biochem J 159: 423–427, 1976.[ISI][Medline]
  55. Tasken K, Skalhegg BS, Tasken KA, Solberg R, Knutsen HK, Levy FO, Sandberg M, Orstavik S, Larsen T, Johansen AK, Vang T, Schrader HP, Reinton NT, Torgersen KM, Hansson V, and Jahnsen T. Structure, function, and regulation of human cAMP-dependent protein kinases. Adv Second Messenger Phosphoprotein Res 31: 191–204, 1997.[ISI][Medline]
  56. Taylor HJ, Chaytor AT, Edwards DH, and Griffith TM. Gap junction-dependent increases in smooth muscle cAMP underpin the EDHF phenomenon in rabbit arteries. Biochem Biophys Res Commun 283: 583–589, 2001.[CrossRef][ISI][Medline]
  57. Taylor SS, Buechler JA, and Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem 59: 971–1005, 1990.[CrossRef][ISI][Medline]
  58. Waldron GJ and Cole WC. Activation of vascular smooth muscle K+ channels by endothelium-derived relaxing factors. Clin Exp Pharmacol Physiol 26: 180–184, 1999.[CrossRef][ISI][Medline]
  59. White R, Bottrill FE, Siau D, and Hiley CR. Protein kinase A-dependent and -independent effects of isoproterenol in rat isolated mesenteric artery: interactions with levcromakalim. J Pharmacol Exp Ther 298: 917–924, 2001.[Abstract/Free Full Text]
  60. White RE, Kryman JP, El-Mowafy AM, Han G, and Carrier GO. cAMP-dependent vasodilators cross-activate the cGMP-dependent protein kinase to stimulate BKCa channel activity in coronary artery smooth muscle cells. Circ Res 86: 897–905, 2000.[Abstract/Free Full Text]
  61. Wichelhaus A, Russ M, Petersen S, and Eckel J. G protein expression and adenylate cyclase regulation in ventricular cardiomyocytes from STZ-diabetic rats. Am J Physiol Heart Circ Physiol 267: H548–H555, 1994.[Abstract/Free Full Text]
  62. Wigg SJ, Tare M, Tonta MA, O'Brien RC, Meredith IT, and Parkington HC. Comparison of effects of diabetes mellitus on an EDHF-dependent and an EDHF-independent artery. Am J Physiol Heart Circ Physiol 281: H232–H240, 2001.[Abstract/Free Full Text]



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