Effect of SMTP-7 on Cisplatin-Induced Nephrotoxicity in Mice.

SMTP-7, a fungal metabolite, is reported to have a high degree of availability for the ischemia-reperfusion (IR)-induced acute kidney injury (AKI) model. Cisplatin, a widely used anticancer drug, has serious side effects, such as AKI. Hence, we aimed to examine the effect of SMTP-7 on cisplatin-induced AKI in this study. Significant increases in blood urea nitrogen (BUN) and serum creatinine (Scr) were observed at 72 h after the intravenous infusion of cisplatin (20 mg/kg). Histologically, necrosis and dilatation (hyaline casts) as well as regeneration were observed in proximal tubules. SMTP-7 inhibited the elevation on BUN and Scr caused by cisplatin dose dependently. The efficacy of SMTP-7 was notable when the drug was administered on the day after cisplatin treatment, whereas the repeated administration of the drug did not result in an enhanced efficacy. Moreover, 10 mg/kg of SMTP-7 considerably ameliorated tubular necrosis and dilatation. The cisplatin treatment also caused an up-regulation of tumor necrosis factor-α (TNF-α) mRNA expression prior to the elevation of the levels of BUN and Scr. Administration of SMTP-7 (10 mg/kg) at 24 h after the cisplatin infusion alleviated the up-regulation of TNF-α mRNA expression. These findings suggest that SMTP-7 exhibits a renoprotective effect against cisplatin infusion based on the inhibition of the expression of pro-inflammatory cytokines such as TNF-α and may be expected a new effective drug for the treatment of cisplatin-induced AKI.

Potent efficacy of Stachybotrys microspora triprenyl phenol-7, a small molecule having anti-inflammatory and antioxidant activities, in a mouse model of acute kidney injury.

Acute kidney injury (AKI) increases the risk of chronic kidney disease (CKD), complicates existing CKD, and can lead to the end-stage renal disease. However, there are no approved effective therapeutics for AKI. Recent studies have suggested that inflammation and oxidative stress are the primary causes of AKI. We previously reported the potential anti-inflammatory and antioxidant activities of Stachybotrys microspora triprenyl phenol-7 (SMTP-7). The aim of the present study was to evaluate the efficacy of SMTP-7 in AKI model mice. AKI was induced in mice by ischemia of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the removal of right kidney. The efficacy of SMTP-7 was determined by measuring the renal function using urine and serum samples and morphological assessment. For deciphering the mechanism of action of SMTP-7, inflammatory cytokines and oxidative stress in kidney were detected. SMTP-7 (0.01, 0.1, 1, 10 mg/kg) dose-dependently improved the renal function. In addition, it improved the damage to renal tubules and exhibited anti-inflammatory and antioxidant activities in the kidney of AKI mice. These results indicate the potential of SMTP-7 as a medicinal compound for the treatment of AKI.

Acetic acid treatment causes renal inflammation and chronic kidney disease in mice.

We established a novel mouse model of chronic kidney disease (CKD) using acetic acid and compared it with the 5/6-nephrectomized mouse model. In our novel model, significant increases were observed in blood biochemical values and urinary parameters. Moreover, a decrease in creatinine clearance (Ccr) was observed. This model also demonstrated a higher survival rate than the 5/6-nephrectomized model. Observed histological changes in our model included cell infiltration in the renal interstitium, tubular dilation, regenerated tubules, and glomerulosclerosis. Inflammation of the renal interstitium was particularly remarkable. TNF-α, IL-1ß, and ICAM-1 mRNA expression were up-regulated prior to elevation of mean blood pressure and prior to changes in blood biochemical values and urinary parameters. Up-regulation of TGF-ß mRNA and down-regulation of nephrin mRNA were also observed at 12 weeks after acetic acid treatment. However, no correlation between the progression of CKD and the decrease in renal blood flow was observed. Finally, repeated losartan administration attenuated the effects of acetic acid-induced renal injury. Our findings suggest that chronic kidney conditions associated with this model may be triggered by interstitial inflammation. Moreover, we suggest that this model is useful for understanding the pathophysiological mechanisms of CKD, and for evaluating the effects of therapeutic agents.

SMTP-44D improves diabetic neuropathy symptoms in mice through its antioxidant and anti-inflammatory activities.

Diabetic neuropathy (DN) is one of the major complications of diabetes. However, there are few approved effective therapies for painful or insensate DN. Recent studies have implicated oxidative stress and inflammation in the pathogenesis of DN, and suppressing these could be an important therapeutic strategy. We previously reported that Stachybotrys microspora triprenyl phenol-44D (SMTP-44D) exhibits both antioxidant and anti-inflammatory activities. The aim of this study was to evaluate the effects of SMTP-44D in a mouse model of streptozotocin-induced DN. SMTP-44D was administered for 3 weeks after the disease induction, and its effects were evaluated on the basis of mechanical and thermal thresholds, blood flow in the bilateral hind paw, and blood flow and conduction velocity in the sciatic nerve. Furthermore, the levels of inflammatory factors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and malondialdehyde (MDA), in the sciatic nerve were assessed. Neurological degeneration was assessed by measuring myelin thickness and g-ratio in the sciatic nerve. SMTP-44D treatment significantly improved allodynia, hyperalgesia, blood flow, and conduction velocity in DN model mice in a dose-dependent manner. Neurological degeneration was also significantly improved, accompanied by decreased levels of inflammatory factors (TNF-α, 57.8%; IL-1ß, 51.4%; IL-6, 62.8%; and MDA, 40.7% reduction rate against the diabetes mellitus + normal saline group). Thus, SMTP-44D can improve allodynia and hyperalgesia in DN without affecting the body weight and blood glucose levels, which may be due to its antioxidant and anti-inflammatory properties. In conclusion, SMTP-44D could be a potential therapeutic agent for the treatment of DN.

Thrombolytic Therapy for Acute Ischemic Stroke: Past and Future.

BACKGROUND: Thromboembolic ischemic stroke, which is mainly caused by hypertension, as well as plasma dyslipidemia, arterial fibrillation and diabetes, is a leading cause of death in the US and other countries. Numerous clinical trials for thrombolytic drugs, which aimed to pharmacologically dissolve thrombi, were conducted in the 1950s, when the first thrombolytic therapy was performed. METHODS: In this study, we summarize the pathophysiologic features of ischemic stroke, and the history of thrombolytic therapy, and discuss the recent progress that has been made in the ongoing development of thrombolytic drugs. CONCLUSION: Thrombolytic therapy is sometimes accompanied by harmful hemorrhagic insults; accordingly, a window of time wherein therapy can safely be performed has been established for this approach. Several basic and clinical studies are ongoing to develop next-generation thrombolytic drugs to expand the time window.

Evaluation of the effects of a new series of SMTPs in the acetic acid-induced embolic cerebral infarct mouse model.

We reported previously that Stachybotrys microspora triprenyl phenol-7 (SMTP-7) showed potential thrombolytic, anti-inflammatory and anti-oxidant effects that account for its excellent pharmacological activity such as having a wider therapeutic time window than tissue plasminogen activator (t-PA) and a significant protection against hemorrhage. The aim of the present study was to evaluate and compare the effect of a new series of SMTPs in the acetic acid-induced embolic cerebral infarct mouse model. Thrombotic occlusion was produced in mice by inducing the transfer of acetic acid-induced thrombi from the right common carotid artery into the brain. SMTPs were evaluated by their effect on reducing infarct area, neurological score and edema. Furthermore, plasmin formation, anti-inflammatory and anti-oxidant activities were assessed by fibrin zymography, measuring pro-inflammatory gene expression, and thiobarbituric acid reactive substances (TBARS) assay, respectively. Treatment with either SMTP-22 or SMTP-43 (10mg/kg), which have similar plasmin formation, anti-inflammatory and anti-oxidant activities to SMTP-7, resulted in reduced infarct area, neurological score and edema. Coexistence of all these three activities appears to be important for the treatment of embolic infarction because SMTP-6, SMTP-25, and SMTP-44D (10mg/kg), which are each missing at least one of the three functions, were not as effective. Therefore, these results indicate that SMTP-22 and SMTP-43 have potential as medicinal compounds for the treatment of embolic cerebral infarction.

Change in soluble epoxide hydrolase (sEH) during cisplatin-induced acute renal failure in mice.

Cisplatin is one of the most effective chemotherapeutic agents against various types of cancers; however, it is also associated with nephrotoxicity. Recently, it was reported that inflammatory mechanisms play a key role in the development of nephrotoxicity. Epoxyeicosatrienoic acids (EETs) have an anti-inflammatory effect and are metabolized by soluble epoxide hydrolase (sEH: encoded by EPHX2 gene). Here, we determined the change in sEH activity and EPHX2 expression in renal tissue associated with the development of cisplatin-induced nephrotoxicity. Cisplatin administration decreased hydrolase activity accompanied by down-regulation of sEH and EPHX2 expression. The down-regulation occurred prior to the elevation of blood urea nitrogen (BUN) and tumor necrosis factor-α (TNF-α) gene expression or at treatment with low dose cisplatin. In addition, a negative correlation was found between EPHX2 expression and renal thiobarbituric acid reactive substance (TBARS), and edaravone, a radical scavenger, administration did not down-regulate expression of this gene. The results of this study suggest that cisplatin decreased sEH activity through the down-regulation of sEH and EPHX2 expression, and this down-regulation was involved in a negative feedback loop to protect renal tissue from further damage. Thus, sEH is a potential therapeutic target of cisplatin-induced nephrotoxicity.

Two types of overcontraction are involved in intrarenal artery dysfunction in type II diabetic mouse.

Contractile responses in small intrarenal arteries are associated with diabetic nephropathy. However, the mechanisms that induce and maintain altered small vessel contraction are not clearly understood. To further understand intrarenal artery dysfunction in diabetes, phenylephrine (PE)-induced force development was assessed in the intrarenal artery [interlobar artery (ILA)] in control (lean) and type II diabetic (ob/ob) mice. PE-induced dose-dependent force development in the ILA was significantly greater in ob/ob mice than in lean mice (592.8 ± 5.2 and 770.1 ± 12.1 µ/mm tissue, respectively, following administration of 30 µM PE, n = 5). Under high-glucose conditions (twice the normal concentration of glucose), PE-induced force development in the ILA was only enhanced in ob/ob mice (946.0 ± 18.2 µN/mm tissue; n = 5). ILA dysfunction reduces blood flow to the glomerulus and may induce diabetic nephropathy. Basal overcontraction of the ILA in ob/ob mice under normal-glucose conditions was reduced by pretreatment with rottlerin, a calcium-independent protein kinase C (PKCδ) inhibitor. Total PKC activity was also reduced by rottlerin. Under high-glucose conditions, the enhanced ILA contraction in diabetic mice was suppressed by rho A and rho kinase inhibitors. Our results indicate two types of ILA dysfunction in diabetes, as follows: 1) a basal increase in PE-induced contraction under normal-glucose conditions, and 2) extracellular glucose-dependent enhancement of PE-induced contraction. We believe that these dysfunctions are mediated by the activation of the PKCδ and rho A-rho kinase pathways, respectively.

Altered gene expression in an embolic stroke model after thrombolysis with tissue plasminogen activator and Stachybotrys microspora triprenyl phenol-7.

The present study compares gene expression and infarct area in a mouse model of embolic stroke after thrombolysis with t-PA and SMTP-7. Embolic occlusion was induced by transfer of acetic acid-induced embolus into the brain. t-PA or SMTP-7 was administered 3 h after embolization. Changes in gene expression were evaluated using microarray and RT-PCR analysis. To determine the involvement of reactive oxygen species in the response to t-PA, the free radical scavenger edaravone was infused immediately before t-PA administration. The expressions of 459 genes involved in the inflammatory response, cell-to-cell signaling, cell movement, and inflammatory disease were altered by embolic occlusion. Twenty-two of those genes were upregulated after t-PA but not SMTP-7 administration. Differences between the t-PA- and SMTP-7-treated groups in the expression of genes including the proinflammatory genes Il6, Stat3, S100a8, and Mmp9 were confirmed with RT-PCR. Edaravone ameliorated the overexpression of these genes. Our data demonstrate differences in gene expression following treatment with SMTP-7 or t-PA that likely explain the difference in therapeutic time windows of the two drugs. ROS are involved in the overexpression of proinflammatory genes. The wide therapeutic time window may be achieved through an anti-oxidative effect and inhibition of proinflammatory gene overexpression.

Adiponectin enhances calcium dependency of mouse bladder contraction mediated by protein kinase Cα expression.

Adiponectin is an adipose tissue-secreted protein and is a multifunctional adipocytokine. However, the association of adiponectin with bladder contraction has not been investigated. In this study, the adiponectin-sense transgenic mouse (Adip-Sen mouse; age, 16-24 weeks; male) and age-matched controls (C57Bl mouse) were studied. The Adip-Sen mouse showed a significant increase in plasma adiponectin levels (56.2%; P < 0.01), compared with those in the C57Bl mouse, without affecting other lipid parameters. Isometric force development in bladder smooth muscle tissues were detected using an organ-bath system. Although carbachol (CCh)-induced (0.1-100 µM) time- and dose-dependent contractions in Adip-Sen mouse bladder were slightly enhanced, compared with those in the C57Bl mouse during a low range (0.3-1.0 µM) of CCh, differences could not be detected with other CCh concentrations. However, the reduction in contraction under Ca(2+)-replaced conditions was significantly different between Adip-Sen and C57Bl mice (94.1 and 66.3% of normal contraction, respectively; n = 5). A parameter of Ca(2+) sensitivity, the relation between intracellular Ca(2+) concentration and contraction, was increased in the Adip-Sen mouse, compared with that in the C57B1 mouse. This Ca(2+) dependency in the Adip-Sen mouse was reduced by a protein kinase C (PKC) inhibitor, but not by a Rho kinase inhibitor. Expression of the calcium-dependent isoform of PKC, PKCα, was increased in the Adip-Sen mouse bladder, and CCh-induced phosphorylation of PKCα was also enhanced, compared with those in the C57Bl mouse. In conclusion, adiponectin is associated with bladder smooth muscle contraction, which involves an increase in Ca(2+) dependency of contraction mediated by PKCα expression.

Two distinct dysfunctions in diabetic mouse mesenteric artery contraction are caused by changes in the Rho A-Rho kinase signaling pathway.

Diabetic complications are associated with small artery dysfunctions. The objective of this study was to identify differences in endothelial cell-denuded mesenteric artery second branch (mesenteric artery-2) contraction, as a typical small artery, between diabetic and non-diabetic mice. Contractile responses in mesenteric artery-2 were assessed in male type 2 diabetic ob/ob mice aged 16-22 weeks and in age-matched control (Lean) mice. Phenylephrine induced dose-dependent contractions in Lean mice (1126.8 ± 28.6 mN/mm tissue at 10 µM phenylephrine; n=5), which were significantly reduced in ob/ob mice (716.8 ± 40.8 mN/mm at 10 µM phenylephrine; n=5). Exposure to high glucose (HG; twice the normal glucose [NG] concentration) enhanced phenylephrine-induced contraction in Lean (1341.4 ± 15.5 mN/mm; n=5) but not in ob/ob mice. These dysfunctions did not involve α(1)-receptor sensitization or protein kinase activity, although the calcium sensitivity of contraction was decreased in ob/ob mice. The Rho kinase inhibitor Y27632 suppressed the difference between Lean and ob/ob mice under NG conditions, which was accompanied by Rho A inactivation. Under HG conditions, glucose-dependent Rho A activation persisted in ob/ob mice whereas Rho kinase expression was reduced. These data suggest that inactivation of Rho A reduced contractibility under NG conditions, and the lack of glucose dependency is associated with reduced Rho kinase expression.

Spatiotemporal dynamics of intracellular calcium in the middle cerebral artery isolated from stroke-prone spontaneously hypertensive rats.

The spatiotemporal dynamics of intracellular calcium within the middle cerebral artery (MCA) isolated from stroke-prone spontaneously hypertensive rats (SHR-SP) were investigated using real-time confocal laser microscopy. At 3 months of age (prestroke), rhythmical changes in the [Ca(2+)](i) during the tonic phase were found to precede vasomotion following application of 5-HT, but not other stimuli. These responses were not observed at 1 month of age; moreover, the MCA lost both responses post-stroke (5 months of age). When [Ca(2+)](i) was analysed in arteriolar smooth muscle cells, rhythmical changes in [Ca(2+)](i) occurred during the same cycle. Thus, these processes were synchronized. The synchronized rhythmical changes in [Ca(2+)](i) were abolished following application of 100 nM ketanserin and 10 µM nicardipine. Treatment with 60 nM charybdotoxin and 10 µM cyclopiazonic acid also significantly reduced rhythmical elevations in [Ca(2+)](i). In addition, rhythmical changes in [Ca(2+)](i) became unsynchronized following treatment with 100 µM carbenoxolone, a gap junction blocker. Connexin 45 mRNA and protein expression were both elevated in the MCA of SHR-SP. Taken together, these findings suggest that rhythmical changes in [Ca(2+)](i) of the MCA are dependent upon the 5-HT(2) receptor-mediated release of calcium from intracellular stores which, in turn, activates voltage-dependent calcium channels to enable an influx of calcium into smooth muscle cells. Subsequently, charybdotoxin-sensitive potassium channels are activated and provide a negative feedback pathway to regulate [Ca(2+)](i). Moreover, the co-ordinated synchronization of rhythmical changes in [Ca(2+)](i) across smooth muscle cells was found to be dependent upon gap junctions.

Neuroprotective mechanisms of SMTP-7 in cerebral infarction model in mice.

Reactive oxygen species (ROS) formation has been found to induce the brain damage following stroke-like events. The aim of the present study was to investigate the effect of Stachybotrys microspora triprenyl phenol-7 (SMTP-7) on the generation of ROS in ischemia-induced cerebral infarction model and in vitro lipid peroxidation. We used immunohistochemistry and real-time reverse-transcription PCR for ex vivo evaluation and thiobarbituric acid-reactive substance reagent assay for in vitro evaluation. We demonstrated that SMTP-7 did not induce enhancement of 4-hydroxynonenal or neutrophil cytosolic factor 2 like t-PA administration at 3 h after ischemia ex vivo and reduce lipid peroxidation in vitro. This compound is the first low molecular weight compound with triplet activities of thrombolytic, anti-inflammatory, and antioxidant activities. We theorized that SMTP-7 is among the pharmacological agents that reduce ROS formation and have been found to limit the extent of brain damage following stroke-like events.

Distinct effects of tissue-type plasminogen activator and SMTP-7 on cerebrovascular inflammation following thrombolytic reperfusion.

BACKGROUND AND PURPOSE: Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. METHODS: The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. RESULTS: Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1ß, TNF-α, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 µg/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. CONCLUSIONS: Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.

A novel finding of a low-molecular-weight compound, SMTP-7, having thrombolytic and anti-inflammatory effects in cerebral infarction of mice.

Tissue plasminogen activator (t-PA) has a short therapeutic time window for administration (3 h) and carries a risk of promoting intracerebral hemorrhage. The aim of the present study was to investigate a therapeutic time window and frequency of hemorrhagic region by treatment with Stachybotrys microspora triprenyl phenol-7 (SMTP-7). Thrombotic occlusion was induced by transfer of acetic acid-induced thrombus at the right common carotid artery into the brain of mice. Infarction area, neurological score, edema percentage, and regional cerebral blood flow (CBF) were determined as the index of the efficacy of SMTP-7. In order to evaluate the mechanism of SMTP-7, plasmin activities and the expressions of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6 mRNA were examined. SMTP-7 (0.1, 1, 10 mg/kg) dose dependently reduced infarction area, neurological score, and edema percentage. Additionally, its therapeutic time window was longer than that of t-PA, a high-molecular-weight compound. In addition, little hemorrhagic region was induced by treatment with SMTP-7. SMTP-7 showed plasmin activity in vivo and caused a decreased CBF to recover. Furthermore, the expressions of inflammatory cytokine mRNA (IL-1beta, TNF-alpha, IL-6) were increased by t-PA treatment 3 h after ischemia but were not induced by SMTP-7 treatment. These results indicate that SMTP-7 shows potential thrombolytic and anti-inflammatory effects as well as a wide therapeutic time window and little hemorrhagic region compared with that of t-PA. Therefore, this novel low-molecular-weight compound may represent a novel approach for the treatment of cerebral infarction.

A novel embolic model of cerebral infarction and evaluation of Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a novel fungal triprenyl phenol metabolite.

The aim of the present study was to establish a novel embolic model of cerebral infarction and to evaluate the effect of Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a novel fungal triprenyl phenol metabolite. Thrombotic occlusion was induced by transfer of acetic acid-induced embolus into the brain. The regional cerebral blood flow was measured by a laser Doppler flowmeter to check the ischemic condition. Infarction area was assessed by 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining. Neurological scores were determined by a modified version of the method described by Longa et al. Emboli were accumulated at the temporal or parietal region of the middle cerebral artery. Additionally, we found that this model showed decreased cerebral blood flow and increased infarction area and neurological scores. Treatment with tissue plasminogen activator (t-PA) reduced infarction area and the neurological scores in a dose-dependent manner; moreover, the decreased cerebral blood flow recovered. SMTP-7 also reduced these values. The therapeutic time window of SMTP-7 was longer than that of t-PA. These results indicate that this model may be useful for understanding the pathophysiological mechanisms of cerebral infarction and evaluating the effects of therapeutic agents. Additionally, SMTP-7 is a promising approach to extend the therapeutic time window. Therefore, this novel compound may represent a novel approach for the treatment of cerebral infarction.

Comparative study of calcium ion dynamics and contractile response in rat middle cerebral and basilar arteries.

The objective of this study was to compare intracellular calcium concentration ([Ca(2+)](i)) and contractile responses in isolated rat middle cerebral artery (MCA) with those in basilar artery (BA) employing real-time confocal laser microscopy. KCl elicited transient [Ca(2+)](i) elevation and sustained contraction in both arteries; moreover, nearly equal responses were evident in both arteries. Application of 5-hydroxytryptamine (5-HT), vasopressin (VP), and alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-me ATP) also induced elevation of [Ca(2+)](i) and contraction in both arteries. The maximum response of 5-HT and VP necessary to increase [Ca(2+)](i) and to constrict the MCA was less in comparison to the BA; however, a linear relationship emerged between the maximum response of [Ca(2+)](i) and that of contraction. Additionally, the slope of the correlation regression line of MCA was nearly identical to that of BA. On the other hand, cyclopiazonic acid (CPA)-induced Ca(2+) release from store sites following contraction of MCA was distinct from that of BA. In MCA, velocity of [Ca(2+)](i) elevation in smooth muscle cells and Ca(2+)-wave propagation along smooth muscle cells induced by 5-HT were slower than those in BA. These observations revealed that different regions of arteries along the same cerebral tissue may display distinct [Ca(2+)](i) response; moreover, this difference may be one reason for the distinct contractile response.

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/mm(2) after 30 microM 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/mm(2); 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.

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 microN/mm(2) following 10 microM 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 microM 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.

A novel approach for the determination of contractile and calcium responses of the basilar artery employing real-time confocal laser microscopy.

INTRODUCTION: Intracellular calcium concentration ([Ca(2+)](i)) modifications in endothelial and smooth muscle cells represent a key element in the pathogenesis of cerebral artery vasospasm. Therefore, the present study documented potential application of confocal laser microscopy in the determination of contractile and [Ca(2+)](i) responses in basilar artery. METHODS: Experiments were performed on the rat isolated basilar artery. Changes in [Ca(2+)](i) were determined by ratiometry involving Fluo-4/AM and Fura Red/AM. Contractile function was calculated from the change in fluorescent area by Fluo-4/AM. RESULTS: KCl (50 mM) elicited an increase in [Ca(2+)](i) and contraction in basilar artery; moreover, nearly well maintained responses were evident for at least 120 min following the first application. 10 microM 5-hydroxytryptamine (5-HT), 10 microM alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP) and 10 nM vasopressin (VP) also induced increases in [Ca(2+)](i) and contraction dose-dependently. Additionally, 10 microM acetylcholine elicited a transient [Ca(2+)](i) decrease and sustained relaxation. In individual cells, rhythmical changes in [Ca(2+)](i) were observed after 10 microM 5-HT. VP (10 nM) evoked modest Ca(2+) oscillation in individual cells; however, Ca(2+) oscillation was not detectable with 10 microM alpha,beta-meATP. DISCUSSION: These results indicate that this method offers reproducibility and quantifiable effects. Imaging technology may therefore be applied to the estimation of [Ca(2+)](i) responses at the tissue level as well as at the level of the individual cell. Thus, confocal laser microscopy is a suitable tool for estimation of small artery function.