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)-1β, tumor necrosis factor-α (TNF-α), 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-1β, TNF-α, 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.

Abstract. 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.

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. Inhibition of SERCA with cyclopiazonic acid (CPA) abolished these differences between WT and PLB‐KO bladder, localizing the effects to the SR. 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. 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. 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.

Abstract-Alterations of the Ca 2ϩ sensitivity of contraction have been reported for porcine coronary artery, but the mechanisms are not clearly understood. We investigated the mechanism(s) of Ca 2ϩ sensitization in response to the thromboxane A 2 analogue (U46619). Our hypothesis is that different mechanisms of Ca 2ϩ sensitization could be distinguished by their distinct time courses. Therefore, we measured the time course of [Ca 2ϩ ] i and isometric force simultaneously in an intact artery after a single addition of U46619. The initial transient phase was associated with Ca 2ϩ release from the sarcoplasmic reticulum, whereas the maintained phase was associated with Ca 2ϩ influx. Two distinct types of Ca 2ϩ sensitization characterized these phases with either protein kinase C (PKC)-mediated or Rho-kinasemediated mechanisms. Their effects were quite distinct on the basis of the time courses over which the sensitization was effective. PKC inhibition (1 mol/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 2ϩ ] i . There were limited effects on the sustained force. Rho-kinase inhibition (10 mol/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 2ϩ ] i relations for KCl contractures. Our evidence suggests that both PKC-mediated and Rho-kinase-mediated Ca 2ϩ sensitizations are present in coronary arteries, but the latter is dominant in thromboxane A 2 receptor-mediated contraction.

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.