Intra- and extrarenal arteries exhibit different profiles of contractile responses in high glucose conditions.

BACKGROUND AND PURPOSE: The renal artery (RA) has been extensively investigated for the assessment of renal vascular function/dysfunction; however, few studies have focused on the intrarenal vasculature. EXPERIMENTAL APPROACH: We devised a microvascular force measurement system, which allowed us to measure contractions of interlobar arteries (ILA), isolated from within the mouse kidney and prepared without endothelium. KEY RESULTS: KCl (50 mM) induced similar force development in the aorta and RA but responses in the ILA were about 50% lower. Treatment of RA with 10 microM phenylephrine (PE), 10 nM U46619 (thromboxane A(2) analogue) or 10 microM prostaglandin F(2 alpha) elicited a response greater than 150% of that induced by KCl. In ILA, 10 nM U46619 elicited a response that was 130% of the KCl-induced response; however, other agonists induced levels similar to that induced by KCl. High glucose conditions (22.2 mM glucose) significantly enhanced responses in RA and ILA to PE or U46619 stimulation. This enhancement was suppressed by rottlerin, a calcium-independent PKC inhibitor, indicating that glucose-dependent, enhanced small vessel contractility in the kidney was linked to the activation of calcium-independent PKC. CONCLUSION AND IMPLICATIONS: Extra- and intrarenal arteries exhibit different profiles of agonist-induced contractions. In ILA, only U46619 enhanced small vessel contractility in the kidney, which might lead to renal dysfunction and nephropathy through reduced intrarenal blood flow rate. A model has been established, which will allow the assessment of contractile responses of intrarenal arteries from murine models of renal disease, including type 2 diabetes.

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 alpha-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.

Distinct pathways of Ca(2+) sensitization in porcine coronary artery: effects of Rho-related kinase and protein kinase C inhibition on force and intracellular Ca(2+).

Alterations of the Ca(2+) sensitivity of contraction have been reported for porcine coronary artery, but the mechanisms are not clearly understood. We investigated the mechanism(s) of Ca(2+) sensitization in response to the thromboxane A(2) analogue (U46619). Our hypothesis is that different mechanisms of Ca(2+) sensitization could be distinguished by their distinct time courses. Therefore, we measured the time course of [Ca(2+)](i) and isometric force simultaneously in an intact artery after a single addition of U46619. The initial transient phase was associated with Ca(2+) release from the sarcoplasmic reticulum, whereas the maintained phase was associated with Ca(2+) influx. Two distinct types of Ca(2+) sensitization characterized these phases with either protein kinase C (PKC)-mediated or Rho-kinase-mediated mechanisms. Their effects were quite distinct on the basis of the time courses over which the sensitization was effective. PKC inhibition (1 micromol/L calphostin C) had a much greater effect in the initial phase, diminishing the size of the transient and prolonging the rise in force and the decline in [Ca(2+)](i). There were limited effects on the sustained force. Rho-kinase inhibition (10 micromol/L Y27632), in contrast, nearly abolished the sustained force but had a lesser effect on the transient phase. Neither inhibitor had any effect on the force versus [Ca(2+)](i) relations for KCl contractures. Our evidence suggests that both PKC-mediated and Rho-kinase-mediated Ca(2+) sensitizations are present in coronary arteries, but the latter is dominant in thromboxane A(2) receptor-mediated contraction.

Preferential role of intracellular Ca2+ stores in regulation of isometric force in NIH 3T3 fibroblast fibres.

Fibroblast contraction plays a major role in wound repair, but the regulatory mechanisms are not well known. We investigated the relations between isometric force and intracellular calcium concentration ([Ca2+]i) in fibroblast fibres. These fibres were made with mouse NIH 3T3 fibroblasts cultured with native collagen in a three-dimensional matrix. Calf serum (CS; 30%) elicited a monotonic increase in force that attained a maximum within 15 min and could be sustained indefinitely. In contrast, [Ca2+]i increased to a peak at 3 min after CS stimulation, then returned to baseline levels by 10 min. Pretreatment with Ca2+-free medium or the Ca2+-channel antagonist nicardipine (10 microM) blocked the CS-induced [Ca2+]i increase, but force was not affected. KCl (50 mM) stimulation on the other hand, elicited a prolonged increase in [Ca2+]i but did not increase force. Inhibition of the endoplasmic reticulum Ca2+ release with Ca2+-ATPase inhibitors cyclopiazonic acid (5 microM) or thapsigargin (5 microM) nearly abolished (<20% control) the increase in [Ca2+]i and force response to CS. Treatment with ryanodine (10 microM) and caffeine (20 mM) had a similar effect. The phospholipase C inhibitor U73122 (3 microM) reduced the CS-induced increases in [Ca2+]i and force by 70 and 40%, respectively. We conclude that fibroblast isometric force is not coupled to Ca2+ arising from transmembrane influx but is correlated with the transient [Ca2+]i increase due to release from intracellular stores. Store-released Ca2+ may initiate activation pathways for fibroblast force development, but is not required for force maintenance.

Effects of microtubules and microfilaments on [Ca(2+)](i) and contractility in a reconstituted fibroblast fiber.

We used a reconstituted fiber formed when 3T3 fibroblasts are grown in collagen to characterize nonmuscle contractility and Ca(2+) signaling. Calf serum (CS) and thrombin elicited reversible contractures repeatable for >8 h. CS elicited dose-dependent increases in isometric force; 30% produced the largest forces of 106 +/- 12 microN (n = 30), which is estimated to be 0.5 mN/mm(2) cell cross-sectional area. Half times for contraction and relaxation were 4.7 +/- 0.3 and 3.1 +/- 0.3 min at 37 degrees C. With imposition of constant shortening velocities, force declined with time, yielding time-dependent force-velocity relations. Forces at 5 s fit the hyperbolic Hill equation; maximum velocity (V(max)) was 0.035 +/- 0. 002 L(o)/s. Compliance averaged 0.0076 +/- 0.0006 L(o)/F(o). Disruption of microtubules with nocodazole in a CS-contracted fiber had no net effects on force, V(max), or stiffness; force increased in 8, but decreased in 13, fibers. Nocodazole did not affect baseline intracellular Ca(2+) concentration ([Ca(2+)](i)) but reduced ( approximately 30%) the [Ca(2+)](i) response to CS. The force after nocodazole treatment was the primary determinant of stiffness and V(max), suggesting that microtubules were not a major component of fiber internal mechanical resistance. Cytochalasin D had major inhibitory effects on all contractile parameters measured but little effect on [Ca(2+)](i).

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.

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.

Receptor-mediated diacylglycerol kinase translocation dependent on both transient increase in the intracellular calcium concentration and modification by protein kinase C.

Diacylglycerol kinase (DG kinase) is activated by various stimuli in many types of cells. We reported earlier that carbachol (CCh) induced DG kinase translocation from the cytosolic fraction to the membrane fraction in guinea pig taenia coli (Biochem. Pharmacol., 50: 591-599, 1995). In this study, the regulation mechanisms of DG kinase translocation are reported, based on the following findings: 1) CCh sustained an increase in DG kinase in the membrane fraction and a decrease in the cytosolic fraction; 2) blocking calcium influx by removing extracellular calcium did not affect the CCh-induced sustained DG kinase translocation; 3) exposing purified protein kinase C (PKC) to DG kinase increased DG kinase affinity to octylglycoside micelles only with the enzyme extracted from the cytosolic fraction; and 4) CCh-induced DG kinase translocation was reversed by removing CCh, and the serine/threonine phosphatase inhibitor, okadaic acid, blocked the reversal of the translocation. These results suggest that CCh-induced DG kinase translocation is promoted by both a transient increase in intracellular calcium, which may be released from the intracellular store, and by DG kinase phosphorylation by PKC.

Protein kinase C is involved in translocation of diacyglycerol 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.

Sensitizing effect of lysophosphatidic acid on mechanoreceptor-linked response in cytosolic free Ca2+ concentration in cultured smooth muscle cells.

We found that lysophosphatidic acid (LPA) sensitizes response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stimulation in cultured longitudinal muscle cells from guinea pig ileum. [Ca2+]i was transiently increased by spritzing of bath solution onto cells as mechanical stimulation in the presence of LPA, but not in absence of LPA. The effect was reversible and concentration-dependent (1-30 nM). Ga3+ but not nicardipine inhibited the [Ca2+]i transient in the presence of LPA. Phosphatidic acid also induced the sensitization, but the effective concentration was more than 10 times higher than in LPA. Histamine and carbachol did not have any sensitizing effect to mechanical stimulation. These results show that LPA sensitizes mechanoreceptor-linked response, suggesting that LPA may play an important role in mechanotransduction mechanisms as an endogenous regulatory factor.

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.

Effect of diacylglycerol kinase inhibitor, R59022 on cytosolic free calcium level and force development in guinea pig taenia coli.

We previously described that carbachol (CCh) increased intracellular phosphatidic acid (PA) in guinea pig taenia coli and the increase was inhibited by diacylglycerol (DG) kinase inhibitor, R59022. The present study was conducted to clarify the role of DG kinase in CCh-induced increase in cytosolic free calcium and force development. Application of CCh to taenia coli induced transient increase in calcium level and contraction followed by sustained and lower calcium level and smaller contraction, and R59022 inhibited the latter responses specifically. The sustained responses were also evoked by 50 mM KCl, but these responses were slightly inhibited by R59022. Inhibition of calcium influx reduced responses to CCh but not KCl. Therefore, we consider that the sustained contraction induced by CCh is attributed to activation of DG kinase which generates PA and accelerates calcium influx.

Role of phosphatidic acid in carbachol-induced contraction in guinea pig Taenia coli.

Carbachol (CCh, 10(-8)-10(-4) M) increased in concentration-dependent manner the mass of phosphatidic acid (PA), but not the mass of diacylglycerol (DG) in the Taenia coli from guinea pig. The increase in the amount of PA caused by CCh was maintained for 20 min. Release of choline from choline phospholipids labeled with [methyl-3H]choline was not changed by CCh. A DG kinase inhibitor, R59022, inhibited the CCh-induced increase in the mass of PA. These results indicate that the increase in the mass of PA by CCh is due to immediate phosphorylation by DG kinase to PA, of the DG produced by phospholipase C (PLC). It is not due to formation of PA by the direct action of phospholipase D. CCh increased 45Ca2+ uptake into the tissue. R59022 inhibited the sustained phase of CCh-induced contraction and 45Ca2+ uptake into the tissue, but only slightly inhibited the initial phase of the CCh-induced contraction. This inhibition by R59022 may result from the inhibitory effect on the CCh-induced increase in PA. These results suggest that CCh activates both PLC and DG kinase and the resultant increase in the mass of PA contributes to the regulation of the sustained phase of CCh-induced contraction which is related to Ca2+ influx.