Koji Nobe

Phospholamban regulation of bladder contractility: evidence from gene-altered mouse models

1 Phospholamban (PLB) is an inhibitor of the sarcoplasmic reticulum (SR) Ca2+‐ATPase (SERCA). Its presence and/or functional significance in contractility of bladder, a smooth muscle tissue particularly dependent on SR function, is unknown. We investigated this by measuring the effects of carbachol (CCh) on force and [Ca2+]i in bladder from mice in which the PLB gene was ablated (PLB‐KO mice). In the PLB‐KO bladder, the maximum increases in [Ca2+]i and force were significantly decreased (41.5 and 47.4 % of WT), and the EC50 values increased. 2 Inhibition of SERCA with cyclopiazonic acid (CPA) abolished these differences between WT and PLB‐KO bladder, localizing the effects to the SR. 3 To determine whether these effects were specific to PLB, we generated mice with smooth‐muscle‐specific expression of PLB (PLB‐SMOE mice), using the SMP8 α‐actin promoter. Western blot analysis of PLB‐SMOE mice showed approximately an eightfold overexpression of PLB while SERCA was downregulated 12‐fold. 4 In PLB‐SMOE bladders, in contrast, the response of [Ca2+]i and force to CCh was significantly increased and the EC50 values were decreased. CPA had little affect on the CCh‐induced increases in [Ca2+]i and force in PLB‐SMOE bladder. 5 These results show that alteration of the PLB:SERCA ratio can significantly modulate smooth muscle [Ca2+]i. Importantly, our data show that PLB can play a major role in modulation of bladder contractility.

Glucose-Dependent Enhancement of Diabetic Bladder Contraction Is Associated with a Rho Kinase-Regulated Protein Kinase C Pathway

Urinary bladder dysfunction, which is one of the most common diabetic complications, is associated with alteration of bladder smooth muscle contraction. However, details regarding the responses under high-glucose (HG) conditions in diabetes are poorly understood. The objective of this study was to identify a relationship between extracellular glucose level and bladder smooth muscle contraction in diabetes. Bladder smooth muscle tissues were isolated from spontaneously type II diabetic (ob/ob mouse; 16-20 weeks of age, male) and age-matched control (C57BL mouse) mice. Carbachol (CCh) induced time- and dose-dependent contractions in ob/ob and C57BL mice; however, maximal responses differed significantly (14.34 ± 0.32 and 12.69 ± 0.22 mN/mm2 after 30 μM CCh treatment, respectively; n = 5-8). Pretreatment of bladders under HG conditions (22.2 mM glucose; concentration is twice that of normal glucose for 30 min) led to enhancement of CCh-induced contraction solely in diabetic mice (15.9 ± 0.26 mN/mm2; n = 5). Basal extracellular glucose-dependent enhancement of bladder contraction in diabetes was documented initially in this study. The correlation between intracellular calcium concentration and contraction was enhanced only in the ob/ob mouse. This enhancement of contraction and total protein kinase C (PKC) activity were inhibited by pretreatment with not only a PKC inhibitor (rottlerin) but also with a rho kinase inhibitor, fasudil [1-(5-isoquinolinesulfonyl)homopiperazine HCl]. These reagents also suppressed the differences between ob/ob and C57BL mouse bladder contractions under HG conditions. The data indicated that glucose-dependent enhancement of contraction in diabetic bladder is involved in the activation of the rho kinase and calcium-independent PKC pathways. This dysfunction may contribute to bladder complications such as detrusor overactivity and reduced bladder capacity in diabetes.

Hyper-reactivity of diacylglycerol kinase is involved in the dysfunction of aortic smooth muscle contractility in streptozotocin-induced diabetic rats

Dysfunction of vascular contraction in diabetes has been reported; however, the mechanisms are poorly understood. In this study, calcium sensitization involving increases in contraction in streptozotocin‐induced diabetic rat aorta was detected. We hypothesize that an alteration in the intracellular signalling system plays a role in the dysfunction of vascular contractility in diabetes. Therefore, diacylglycerol (DG) kinase as a key enzyme of phosphatidylinositol (PI) turnover was investigated. Treatment with norepinephrine (NE) caused time‐ and dose‐dependent activation of DG kinase in control rats. This activation required simultaneous increases in intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activation. In diabetic rats, hyper‐reactivity of DG kinase involving inactivation in the resting state and over‐activation in NE stimulation was observed. During hyper‐reactivity, [Ca2+]i dependency of DG kinase was enhanced. Treatment with 50 mM KCl induced significant escalation in activity; moreover, basal activation of PKC was detected only in diabetes. These results suggested that PKC had been activated in the resting state. In contrast, these conditions were insufficient for DG kinase activation due to the absence of [Ca2+]i elevation. During NE‐stimulation, PKC activation was maintained and [Ca2+]i increased. Therefore, DG kinase was activated and an elevation in calcium dependency enhanced this activation. The present study suggested that DG kinase hyper‐reactivity in diabetes involved both an increase in [Ca2+]i and basal activation of PKC. This phenomenon may be associated with increased vascular contraction in diabetes mediated by acceleration of PI‐turnover.

Alternations of Diacylglycerol Kinase in Streptozotocin-Induced Diabetic Rats

Dysfunction of organs has been reported in diabetic rats, suggesting an association with changes in intracellular signal transduction pathways including phosphatidylinositol (PI) turnover. Diacylglycerol (DG) kinase catalyses the phosphorylation of DG, which is considered to play a major physiological role in the metabolism of the intracellular messenger DG. However, no relation between DG kinase activity and any disease in mammalian tissue has been reported to date. In the present study, we investigated whether the changes in DG kinase activity are related to diabetes. Basal resting level of DG kinase activity changed in tissue isolated from diabetic rats. Decreases in resting activity detected in aorta and kidney and agonist-induced responses differed between these tissues. Submaximal increases in basal activity also were detected in vas deferens and hepatocytes. These changes in DG kinase activity resemble the functional changes associated with complications of diabetes, suggesting that changes in PI turnover followed by DG kinase activity are a key element in the complications. It is the first study about the changes in DG kinase activity in mammalian disease.

High-Glucose-Altered Endothelial Cell Function Involves Both Disruption of Cell-to-Cell Connection and Enhancement of Force Development

Vascular endothelial cells (ECs), which regulate vascular tonus, serve as a barrier at the interface of vascular tissue. It is generally believed that alteration of this barrier is correlated with diabetic complications; however, a detailed mechanism has not been elucidated. This study examined alteration of bovine arterial EC functions stimulated by a thromboxane A2 analog (9,11-dideoxy-11α,9α-epoxymethano prostaglandin F2α; U46619) under normal and high-glucose (HG) conditions. U46619 treatment increased EC layer permeability in a time- and dose-dependent fashion. This response initially disrupted calcium-dependent EC-to-EC connections, namely, vascular endothelial cadherin (VE-CaD). Thereafter, EC force development in association with morphological changes was detected employing a reconstituted EC fiber technique, resulting in paracellular hole formation in the EC layer. Thus, we confirmed that U46619-induced enhancement of EC layer permeability involves these sequential steps. Similar trials were performed using a concentration twice that of normal glucose (22.2 mM glucose for 48 h). This treatment significantly enhanced U46619-induced EC layer permeability; furthermore, increases in both rate of VE-CaD disruption and EC fiber contraction were evident. Inhibition of calcium-independent protein kinase C and diacylglycerol kinase indicated that the glucose-dependent increase in VE-CaD disruption was mediated by a calcium-independent mechanism. Moreover, EC contraction was regulated by a typical calcium-independent pathway associated with rho kinase and actin stress fiber. Contraction was also enhanced under HG conditions. This investigation revealed that glucose-dependent enhancement of EC layer permeability is related to increases in VE-CaD disruption and EC contraction. Increases in both parameters were mediated by alteration of a calcium-independent pathway.

Novel diacylglycerol kinase inhibitor selectively suppressed an U46619-induced enhancement of mouse portal vein contraction under high glucose conditions

Diacylglycerol kinase (DG kinase) is a key enzyme in vascular contraction; however, alterations of the regulatory mechanisms in vascular dysfunction are poorly understood. In this study, the effect of a novel DG kinase inhibitor, stemphone, on vascular contraction was investigated. The conventional DG kinase inhibitor, 6‐[2‐(4‐[(4‐fluorophenyl)phenyl‐methylene]‐1‐piperidinyl)ethyl]‐7‐methyl‐5H‐thiazolo [3,2‐α] pyrimidine‐5‐one (R59022) (0.1–30 μM), inhibited thromboxane A2 analogue 9,11‐dideoxy‐11α,9α‐epoxymethanoprostaglandin F2α (U46619)‐induced sustained contractions in mouse aorta and porcine coronary artery in a dose‐dependent manner. Treatment with stemphone did not affect contractions in these tissues. However, stemphone significantly inhibited (>0.3 μM) U46619‐induced spontaneous phasic contraction in mouse portal vein. This inhibitory effect was not detected following R59022 treatment in portal vein. Therefore, stemphone demonstrated selectivity in terms of portal vein contraction. Under high glucose (22.2 mM) conditions, U46619‐induced contraction was enhanced in these three types of vascular tissue. Inhibitory effects of R59022 were attenuated under these conditions; however, effects of stemphone were observed. These results indicated that stemphone could inhibit portal vein contraction under high glucose conditions, for example, diabetes. These data suggested the possibility that DG kinase may be a target of hyperportal pressure. Total mass of DG was enhanced under high glucose conditions. DG was derived from incorporated glucose via de novo synthesis in the absence of phospholipase C pathway mediation. This enhanced DG under high glucose conditions activated a calcium‐independent protein kinase C (PKC). This PKC was associated with calcium‐independent DG kinase activation. Treatment with stemphone also inhibited calcium‐independent DG kinase. These signal transduction pathways were distinguishable from a DG–PKC pathway under normal glucose conditions. The present investigation suggested that stemphone selectively inhibited overcontraction of portal vein induced by high glucose levels. This phenomenon was attributable to inhibition of calcium‐independent DG kinase activation that occurred under high glucose conditions mediated by both DG synthesized from glucose and calcium‐independent PKC activation.

Protein kinase C is involved in translocation of diacylglycerol kinase induced by carbachol in guinea pig taenia coli

The regulatory mechanisms of diacylglycerol (DG) kinase activity were studied in guinea pig taenia coli. In an octylglycoside mixed micellar assay system, DG kinase activities were distributed in both membrane and cytosolic fractions. Treatment of the tissue with carbachol (CCh) increased the activity in the membrane fraction and decreased the cytosolic fraction without affecting total DG kinase activity. The Km value of DG kinase in the membrane fraction was unchanged by treatment with CCh, although Vmax was increased. These findings suggest that DG kinase may be translocated from the cytosol to the membrane by CCh-stimulation. Increase in DG content by treatment of tissue with a cell-permeable species of DG, dioctanoyl-sn-glycerol, did not induce DG kinase translocation. Each treatment with protein kinase C (PKC) inhibitor and PKC-desensitization blocked CCh-induced DG kinase translocation; and phorbol ester induced the translocation only in intracellular calcium-accumulated tissues. Considering these results, CCh-induced DG kinase activation appears to involve DG kinase translocation from the cytosol to the membrane in association with both PKC and intracellular calcium concentration rather than cellular DG content.

Activation of diacylglycerol kinase by carbachol in guinea pig taenia coli

Changes in diacylglycerol kinase (DG kinase) activity in carbachol (CCh)-stimulated guinea pig taenia coli were investigated. In a mixed micellar assay system, added 1,2-dioctanoyl-sn-glycerol (diC8) and endogenous DG were competitively bound to common DG kinase isozymes from guinea pig taenia coli and phosphorylated, suggesting that diC8 is useful as a probe of agonist effects on DG kinase activity. In phosphorus-32 ([32P]Pi)- and diC8-prelabeled guinea pig taenia coli, diC8 was phosphorylated by DG kinase to [32P]dioctanoyl-phosphatidic acid ([32P]diC8-PA). CCh increased the accumulation of both [32P]diC8-PA and endogenous [32P]phosphatidic acid ([32P]PA) in a time- and dose-dependent manner (0.1-100 microM CCh). CCh-induced increases in [32P]diC8-PA and [32P]PA were inhibited by 1 microM atropine and 3 microM DG kinase inhibitor (R59022). These findings indicated the activation of DG kinase by muscarinic receptor stimulation in guinea pig taenia coli. Therefore, DG kinase activation may play an important role in CCh-induced PA formation. CCh-induced [32P]diC8-PA and [32P]PA accumulation was dependent on intracellular calcium concentrations. However, a KCl-induced increase in intracellular calcium, without receptor stimulation, was ineffective. Moreover, treatment with phorbol ester also increased accumulation of both PA species in KCl-treated tissues. These findings suggest that muscarinic receptor mediated activation of DG kinase may require both an increase in intracellular calcium and PKC activation in guinea pig taenia coli.

SUBCELLULAR DISTRIBUTION OF PROTEIN KINASE C ISOFORMS IN GASTRIC ANTRUM SMOOTH MUSCLE OF STZ-INDUCED DIABETIC RATS

Contractile responses to carbachol (CCh), protein kinase C (PKC) activity and distribution of PKC isoforms in subcellular fractions isolated from gastric antrum smooth muscle of control and streptozotocin (STZ)-induced diabetic rats were examined. CCh induced concentration-dependent contraction in antrum smooth muscle from controls and diabetics, and this contraction was significantly greater in diabetics than in controls. In diabetics, the PKC activity in the nucleus fraction was significantly decreased by about 63% in the resting condition and that in the cytosol fraction was significantly increased by about 135% after the treatment with 10 microM CCh for 10 min compared to controls. Immunoblot analysis showed that 8 PKC isoforms (-alpha, -beta, -gamma, -delta, -epsilon, -zeta, -iota, -lambda) were expressed in rat antrum smooth muscle. The PKC-beta isoform was significantly decreased by about 47% in the nucleus fraction in the resting condition in diabetics compared to controls. The nucleus, cytosol and membrane fractions of this isoform were decreased in controls after the treatment with 10 microM CCh for 10 min whereas these fractions were unchanged in diabetics. The PKC-epsilon significantly increased by about 219% in the cytosol fraction of diabetics in the resting condition, but the distribution of this isoform was unchanged in controls and diabetics after the treatment with 10 microM CCh for 10 min. Results suggest that the diversity of PKC isoform-specific distribution and translocation may be related to abnormal contractility and intracellular signal transduction through the PKC pathway in diabetics.

Alterations of Glucose-Dependent and -Independent Bladder Smooth Muscle Contraction in Spontaneously Hypertensive and Hyperlipidemic Rat

Alteration of bladder contractility was examined in the spontaneously hypertensive and hyperlipidemic rat (SHHR; age, 9 months; systolic blood pressure, >150 mm Hg; plasma cholesterol, >150 mg/dl). Carbachol (CCh) induced time- and dose-dependent contractions in Sprague-Dawley (age-matched control) rats and SHHR; however, maximal levels differed significantly (13.3 ± 2.2 and 5.4 ± 1.9 μN/mm2 following 10 μM CCh treatment, respectively; n = 5). This difference, which was maintained in calcium-replaced physiological salt solution (PSS), was suppressed by pretreatment with rho kinase inhibitor, 1 μM Y27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide]; moreover, total activity of rho kinase was also reduced in SHHR bladder. Pretreatment of bladders under high-glucose (HG) conditions (22.2 mM glucose-contained PSS for 30 min) led to enhancement of CCh-induced contraction solely in control animals. Under HG conditions, both protein kinase C (PKC) activity and production of diacylglycerol (DG) derived from incorporated glucose declined in SHHR bladder; however, sustained elevation of plasma glucose level was not detected in SHHR. These results suggested that bladder contractility dysfunction in SHHR is attributable to alteration of rho kinase activity and the DG-PKC pathway. This dysfunction may occur prior to chronic hyperglycemia onset in progressive hypertension and hyperlipidemia.