Tethered platelet capture provides a mechanism for restricting circulating platelet activation to the wound site.

Background: Puncture wounding is a longstanding challenge to human health for which understanding is limited, in part, by a lack of detailed morphological data on how the circulating platelet capture to the vessel matrix leads to sustained, self-limiting platelet accumulation. Objectives: The objective of this study was to produce a paradigm for self-limiting thrombus growth in a mouse jugular vein model. Methods: Data mining of advanced electron microscopy images was performed from authors' laboratories. Results: Wide-area transmission electron mcrographs revealed initial platelet capture to the exposed adventitia resulted in localized patches of degranulated, procoagulant-like platelets. Platelet activation to a procoagulant state was sensitive to dabigatran, a direct-acting PAR receptor inhibitor, but not to cangrelor, a P2Y12 receptor inhibitor. Subsequent thrombus growth was sensitive to both cangrelor and dabigatran and sustained by the capture of discoid platelet strings first to collagen-anchored platelets and later to loosely adherent peripheral platelets. Spatial examination indicated that staged platelet activation resulted in a discoid platelet tethering zone that was pushed progressively outward as platelets converted from one activation state to another. As thrombus growth slowed, discoid platelet recruitment became rare and loosely adherent intravascular platelets failed to convert to tightly adherent platelets. Conclusions: In summary, the data support a model that we term Capture and Activate, in which the initial high platelet activation is directly linked to the exposed adventitia, all subsequent tethering of discoid platelets is to loosely adherent platelets that convert to tightly adherent platelets, and self-limiting, intravascular platelet activation over time is the result of decreased signaling intensity.

Self-paced polling increases medical student engagement in recorded lectures and improves examination performance.

Engaging preclinical medical students in the curriculum is challenging. To address this challenge, the investigators developed and implemented self-paced polling with recorded lectures, in which students answered audience response questions at their own pace. In 2021, we retrospectively assigned second-year medical students (N = 165) as Active or Inactive based on their answered polling questions. We subdivided the Active group into two groups, a Live group who predominantly responded to polling in live classes and a Self-paced group who predominantly used polling with recorded lectures. Outcomes were academic performance on customized National Board of Medical Examiners (NBME) examinations and engagement. Compared with the Inactive group, the Self-paced group performed better on the customized NBME examination after extensive self-paced polling. Students answered a significantly larger proportion of questions correctly in self-paced polling compared with live polling. Students who used self-paced polling reported a positive experience and indicated they had emotional, behavioral, or cognitive engagement with the curriculum. This study introduces self-paced polling with recorded lectures, which medical educators can potentially use to enhance student engagement and academic performance.NEW & NOTEWORTHY More medical students utilize recorded lectures than live lectures. Self-paced polling questions allow students to participate while watching recorded lectures. Second-year medical students performed significantly better on examination after actively using the self-paced polling compared with inactive students. They also reported emotional, behavioral, and cognitive engagement with the course material while using the self-paced polling.

The IFNγ-PDL1 Pathway Enhances CD8T-DCT Interaction to Promote Hypertension.

BACKGROUND: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. METHODS: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8+ T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. RESULTS: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. CONCLUSIONS: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

Interactive retrieval practice in renal physiology improves performance on customized National Board of Medical Examiners examination of medical students.

Retrieval practice improves long-term retention. Use of interactive retrieval practice in large group, in-person and online live classes, in combination with outside resources, is unreported for medical physiology classes. The primary study purpose was to compare student cohorts' performance with or without retrieval practice in renal physiology classes, relative to the national average on customized national examinations in renal physiology, nonphysiology, and all questions. The secondary purpose was to examine the students' educational experience. For the primary purpose, we used a nonequivalent group, posttest-only design. For the secondary purpose, we used cross-sectional and qualitative designs. We analyzed examination results of 684 students in four academic years. For renal physiology questions, students performed significantly better in years with retrieval practice compared with years without it (P < 0.001). There was no change in nonphysiology scores over the four years. Performance in all questions, too, significantly improved (P < 0.001). A large majority (86%) of students indicated retrieval practice helped them learn renal physiology. Student ratings of quality in online classes, which featured interactive retrieval practice, were higher than that of in-person classes (P < 0.001). Qualitative analysis revealed students found interactive retrieval practice, scaffolding, outside resources, and the instructor's teaching style helpful. Educators in medical physiology classes can use our findings to implement interactive retrieval practice.

Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates.

Primary hemostasis results in a platelet-rich thrombus that has long been assumed to form a solid plug. Unexpectedly, our 3-dimensional (3D) electron microscopy of mouse jugular vein puncture wounds revealed that the resulting thrombi were structured about localized, nucleated platelet aggregates, pedestals and columns, that produced a vaulted thrombus capped by extravascular platelet adherence. Pedestal and column surfaces were lined by procoagulant platelets. Furthermore, early steps in thrombus assembly were sensitive to P2Y12 inhibition and late steps to thrombin inhibition. Based on these results, we propose a Cap and Build, puncture wound paradigm that should have translational implications for bleeding control and hemostasis.

Metoprolol Impairs ß1-Adrenergic Receptor-Mediated Vasodilation in Rat Cerebral Arteries: Implications for ß-Blocker Therapy.

The practice of prescribing ß-blockers to lower blood pressure and mitigate perioperative cardiovascular events has been questioned because of reports of an increased risk of stroke. The benefit of ß-blocker therapy primarily relies on preventing activation of cardiac ß1-adrenergic receptors (ARs). However, we reported that ß1ARs also mediate vasodilator responses of rat cerebral arteries (CAs), implying that ß-blockers may impair cerebral blood flow under some conditions. Here, we defined the impact of metoprolol (MET), a widely prescribed ß1AR-selective antagonist, on adrenergic-elicited diameter responses of rat CAs ex vivo and in vivo. MET (1-10 µmol/l) prevented ß1AR-mediated increases in diameter elicited by dobutamine in cannulated rat CAs. The ß1AR-mediated dilation elicited by the endogenous adrenergic agonist norepinephrine (NE) was reversed to a sustained constriction by MET. Acute oral administration of MET (30 mg/kg) to rats in doses that attenuated resting heart rate and dobutamine-induced tachycardia also blunted ß1AR-mediated dilation of CAs. In the same animals, NE-induced dilation of CAs was reversed to sustained constriction. Administration of MET for 2 weeks in drinking water (2 mg/ml) or subcutaneously (15 mg/kg per day) also resulted in NE-induced constriction of CAs in vivo. Thus, doses of MET that protect the heart from adrenergic stimulation also prevent ß1AR-mediated dilation of CAs and favor anomalous adrenergic constriction. Our findings raise the possibility that the increased risk of ischemic stroke in patients on ß-blockers relates in part to adrenergic dysregulation of cerebrovascular tone. SIGNIFICANCE STATEMENT: ß-Blocker therapy using second-generation, cardioselective ß-blockers is associated with an increased risk of stroke, but the responsible mechanisms are unclear. Here, we report that either acute or chronic systemic administration of a cardioselective ß-blocker, metoprolol, mitigates adrenergic stimulation of the heart as an intended beneficial action. However, metoprolol concomitantly eliminates vasodilator responses to adrenergic stimuli of rat cerebral arteries in vivo as a potential cause of dysregulated cerebral blood flow predisposing to ischemic stroke.

Using a Facebook group to facilitate faculty-student interactions during preclinical medical education: a retrospective survey analysis.

BACKGROUND: Strong learner-teacher relationships are associated with more successful learning outcomes. With shortened modular curricula and increased availability of online resources, fostering faculty interaction with preclinical medical students has become more challenging. We sought to enhance learner-teacher relationships by engaging in discussion with preclinical medical students in their own online space. METHODS: We utilized a closed Facebook discussion group, where faculty and students voluntarily joined in informal discussions and shared announcements related to their courses. The closed discussion group allowed only participating students and faculty to see others' posts within the group. This provided a platform to freely interact within the confines of the group while maintaining privacy for the personal Facebook accounts of both faculty and students. We utilized the discussion group through three separate organ system-based modules for 14 weeks. Afterward, students were asked to complete an anonymous, voluntary online survey about their experience. RESULTS: 94.1% (160/170) of enrolled second-year medical students joined the voluntary FB discussion group. There were 214 posts, 628 comments, and 4166 reactions in this discussion group during the three modules. Of the students in the group, 74.4% (119/160) responded to the online survey. Overall, students strongly agreed that the Facebook discussion group fostered better rapport with faculty, helped content learning, and improved emotional well-being. Also, they felt more comfortable seeking academic help after using the discussion group. They reported a slight preference for Facebook over email as a medium for asking questions, but no preference for either as a medium for distributing announcements. Students overwhelmingly recommended that the discussion group should be continued in future years. CONCLUSION: The Facebook discussion group was a free, efficient, and effective method of cultivating the learner-teacher relationship with the preclinical medical students, resulting in reported enhancement of learning and morale.

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 µmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 µmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

Molecular determinants of beta-adrenergic signaling to voltage-gated K+ channels in the cerebral circulation.

Voltage-gated K+ (Kv ) channels are major determinants of membrane potential in vascular smooth muscle cells (VSMCs) and regulate the diameter of small cerebral arteries and arterioles. However, the intracellular structures that govern the expression and function of vascular Kv channels are poorly understood. Scaffolding proteins including postsynaptic density 95 (PSD95) recently were identified in rat cerebral VSMCs. Primarily characterized in neurons, the PSD95 scaffold has more than 50 known binding partners, and it can mediate macromolecular signaling between cell-surface receptors and ion channels. In cerebral arteries, Shaker-type Kv 1 channels appear to associate with the PSD95 molecular scaffold, and PSD95 is required for the normal expression and vasodilator influence of members of this K+ channel gene family. Furthermore, recent findings suggest that the ß1-subtype adrenergic receptor is expressed in cerebral VSMCs and forms a functional vasodilator complex with Kv 1 channels on the PSD95 scaffold. Activation of ß1-subtype adrenergic receptors in VSMCs enables protein kinase A-dependent phosphorylation and opening of Kv 1 channels in the PSD95 complex; the subsequent K+ efflux mediates membrane hyperpolarization and vasodilation of small cerebral arteries. Early evidence from other studies suggests that other families of Kv channels and scaffolding proteins are expressed in VSMCs. Future investigations into these macromolecular complexes that modulate the expression and function of Kv channels may reveal unknown signaling cascades that regulate VSMC excitability and provide novel targets for ion channel-based medications to optimize vascular tone.

CD8+ T cells stimulate Na-Cl co-transporter NCC in distal convoluted tubules leading to salt-sensitive hypertension.

Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention and salt-sensitive hypertension is unknown. Here we demonstrate that T cells infiltrate into the kidney of salt-sensitive hypertensive animals. In particular, CD8+ T cells directly contact the distal convoluted tubule (DCT) in the kidneys of DOCA-salt mice and CD8+ T cell-injected mice, leading to up-regulation of the Na-Cl co-transporter NCC, p-NCC and the development of salt-sensitive hypertension. Co-culture with CD8+ T cells upregulates NCC in mouse DCT cells via ROS-induced activation of Src kinase, up-regulation of the K+ channel Kir4.1, and stimulation of the Cl- channel ClC-K. The last event increases chloride efflux, leading to compensatory chloride influx via NCC activation at the cost of increasing sodium retention. Collectively, these findings provide a mechanism for adaptive immunity involvement in the kidney defect in sodium handling and the pathogenesis of salt-sensitive hypertension.

BK channels in rat and human pulmonary smooth muscle cells are BKα-ß1 functional complexes lacking the oxygen-sensitive stress axis regulated exon insert.

A loss of K+ efflux in pulmonary arterial smooth muscle cells (PASMCs) contributes to abnormal vasoconstriction and PASMC proliferation during pulmonary hypertension (PH). Activation of high-conductance Ca2+-activated (BK) channels represents a therapeutic strategy to restore K+ efflux to the affected PASMCs. However, the properties of BK channels in PASMCs-including sensitivity to BK channel openers (BKCOs)-are poorly defined. The goal of this study was to compare the properties of BK channels between PASMCs of normoxic (N) and chronic hypoxic (CH) rats and then explore key findings in human PASMCs. Polymerase chain reaction results revealed that 94.3% of transcripts encoding BKα pore proteins in PASMCs from N rats represent splice variants lacking the stress axis regulated exon insert, which confers oxygen sensitivity. Subsequent patch-clamp recordings from inside-out (I-O) patches confirmed a dense population of BK channels insensitive to hypoxia. The BK channels were highly activated by intracellular Ca2+ and the BKCO lithocholate; these responses require BKα-ß1 subunit coupling. PASMCs of CH rats with established PH exhibited a profound overabundance of the dominant oxygen-insensitive BKα variant. Importantly, human BK (hBK) channels in PASMCs from human donor lungs also represented the oxygen-insensitive BKα variant activated by BKCOs. The hBK channels showed significantly enhanced Ca2+ sensitivity compared with rat BK channels. We conclude that rat BK and hBK channels in PASMCs are oxygen-insensitive BKα-ß1 complexes highly sensitive to Ca2+ and the BKCO lithocholate. BK channels are overexpressed in PASMCs of a rat model of PH and may provide an abundant target for BKCOs designed to restore K+ efflux.

Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

Beta1-adrenergic receptor-mediated dilation of rat cerebral artery requires Shaker-type KV1 channels on PSD95 scaffold.

Postsynaptic density-95 (PSD95) is a scaffolding protein in cerebral vascular smooth muscle cells (cVSMCs), which binds to Shaker-type K(+) (KV1) channels and facilitates channel opening through phosphorylation by protein kinase A. ß1-Adrenergic receptors (ß1ARs) also have a binding motif for PSD95. Functional association of ß1AR with KV1 channels through PSD95 may represent a novel vasodilator complex in cerebral arteries (CA). We explored whether a ß1AR-PSD95-KV1 complex is a determinant of rat CA dilation. RT-PCR and western blots revealed expression of ß1AR in CA. Isoproterenol induced a concentration-dependent dilation of isolated, pressurized rat CA that was blocked by the ß1AR blocker CGP20712. Cranial window imaging of middle cerebral arterioles in situ showed isoproterenol- and norepinephrine-induced dilation that was blunted by ß1AR blockade. Isoproterenol-induced hyperpolarization of cVSMCs in pressurized CA was blocked by CGP20712. Confocal images of cVSMCs immunostained with antibodies against ß1AR and PSD95 indicated strong colocalization, and PSD95 co-immunoprecipitated with ß1AR in CA lysate. Blockade of KV1 channels, ß1AR or disruption of PSD95-KV1 interaction produced similar blunting of isoproterenol-induced dilation in pressurized CA. These findings suggest that PSD95 mediates a vasodilator complex with ß1AR and KV1 channels in cVSMCs. This complex may be critical for proper vasodilation in rat CA.

Protein kinase A-phosphorylated KV1 channels in PSD95 signaling complex contribute to the resting membrane potential and diameter of cerebral arteries.

RATIONALE: Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K(+) (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells of the cerebral (cVSMCs) circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown. OBJECTIVE: We explored whether a specific interaction between PSD95 and KV1 channels enables protein kinase A phosphorylation of KV1 channels in cVSMCs to promote vasodilation. METHODS AND RESULTS: Rat cerebral arteries were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of KV1.2α was designed as a dominant-negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. CONCLUSIONS: These findings provide initial evidence that protein kinase A phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in cerebral arteries and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.

Ion channel remodeling in vascular smooth muscle during hypertension: Implications for novel therapeutic approaches.

Ion channels are multimeric, transmembrane proteins that selectively mediate ion flux across the plasma membrane in a variety of cells including vascular smooth muscle cells (VSMCs). The dynamic interplay of Ca(2+) and K(+) channels on the plasma membrane of VSMCs plays a pivotal role in modulating the vascular tone of small arteries and arterioles. The abnormally-elevated arterial tone observed in hypertension thus points to an aberrant expression and function of Ca(2+) and K(+) channels in the VSMCs. In this short review, we focus on the three well-studied ion channels in VSMCs, namely the L-type Ca(2+) (CaV1.2) channels, the voltage-gated K(+) (KV) channels, and the large-conductance Ca(2+)-activated K(+) (BK) channels. First, we provide a brief overview on the physiological role of vascular CaV1.2, KV and BK channels in regulating arterial tone. Second, we discuss the current understanding of the expression changes and regulation of CaV1.2, KV and BK channels in the vasculature during hypertension. Third, based on available proof-of-concept studies, we describe the potential therapeutic approaches targeting these vascular ion channels in order to restore blood pressure to normotensive levels.

Canonical transient receptor channel 5 (TRPC5) and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms.

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.

The ß3 subunit contributes to vascular calcium channel upregulation and hypertension in angiotensin II-infused C57BL/6 mice.

Voltage-gated L-type Ca(2+) (Ca(v)1.2) channels in vascular smooth muscle cells are a predominant Ca(2+) influx pathway that mediates arterial tone. Channel biogenesis is accomplished when the pore-forming α(1C) subunit coassembles with regulatory Ca(v)ß subunits intracellularly, and the multiprotein Ca(v)1.2 channel complex translocates to the plasma membrane to form functional Ca(2+) channels. We hypothesized that the main Ca(v)ß isoform in vascular smooth muscle cells, Ca(v)ß3, is required for the upregulation of arterial Ca(v)1.2 channels during the development of hypertension, an event associated with abnormal Ca(2+)-dependent tone. Ca(v)1.2 channel expression and function were compared between second-order mesenteric arteries of C57BL/6 wild-type (WT) and Ca(v)ß3(-/-) mice infused with saline (control) or angiotensin II (Ang II) for 2 weeks to induce hypertension. The mesenteric arteries of Ang II-infused WT mice showed increased Ca(v)1.2 channel expression and accentuated Ca(2+)-mediated contractions compared with saline-infused WT mice. In contrast, Ca(v)1.2 channels failed to upregulate in mesenteric arteries of Ang II-infused Ca(v)ß3(-/-) mice, and Ca(2+)-dependent reactivity was normal in these arteries. Basal systolic blood pressure was not significantly different between WT and Ca(v)ß3(-/-) mice (98 ± 2 and 102 ± 3 mm Hg, respectively), but the Ca(v)ß3(-/-) mice showed a blunted pressor response to Ang II infusion. Two weeks after the start of Ang II administration, the systolic blood pressure of Ca(v)ß3(-/-) mice averaged 149 ± 4 mm Hg compared with 180 ± 5 mm Hg in WT mice. Thus, the Ca(v)ß3 subunit is a critical regulatory protein required to upregulate arterial Ca(v)1.2 channels and fully develop Ang II-dependent hypertension in C57BL/6 mice.

Postsynaptic density-95 scaffolding of Shaker-type K⁺ channels in smooth muscle cells regulates the diameter of cerebral arteries.

Postsynaptic density-95 (PSD95) is a 95 kDa scaffolding molecule in the brain that clusters postsynaptic proteins including ion channels, receptors, enzymes and other signalling partners required for normal cognition. The voltage-gated, Shaker-type K(+) (K(V)1) channel is one key binding partner of PSD95 scaffolds in neurons. However, K(V)1 channels composed of α1.2 and α1.5 pore-forming subunits also are expressed in the vascular smooth muscle cells (cVSMCs) of the cerebral circulation, although the identity of their molecular scaffolds is unknown. Since α1.2 contains a binding motif for PSD95, we explored the possibility that cVSMCs express PSD95 as a scaffold to promote K(V)1 channel expression and cerebral vasodilatation. Cerebral arteries from Sprague-Dawley rats were isolated for analysis of PSD95 and K(V)1 channel proteins. PSD95 was detected in cVSMCs and it co-immunoprecipitated and co-localized with the pore-forming α1.2 subunit of the K(V)1 channel. Antisense-mediated knockdown of PSD95 profoundly reduced K(V)1 channel expression and suppressed K(V)1 current in patch-clamped cVSMCs. Loss of PSD95 also depolarized cVSMCs in pressurized cerebral arteries and induced a strong constriction associated with a loss of functional K(V)1 channels. Our findings provide initial evidence that PSD95 is expressed in cVSMCs, and the K(V)1 channel is one of its important binding partners. PSD95 appears to function as a critical 'dilator' scaffold in cerebral arteries by increasing the number of functional K(V)1 channels at the plasma membrane.

Hemodynamic changes in the kidney in a pediatric rat model of sepsis-induced acute kidney injury.

Sepsis is a leading cause of acute kidney injury (AKI) and mortality in children. Understanding the development of pediatric sepsis and its effects on the kidney are critical in uncovering new therapies. The goal of this study was to characterize the development of sepsis-induced AKI in the clinically relevant cecal ligation and puncture (CLP) model of peritonitis in rat pups 17-18 days old. CLP produced severe sepsis demonstrated by time-dependent increase in serum cytokines, NO, markers of multiorgan injury, and renal microcirculatory hypoperfusion. Although blood pressure and heart rate remained unchanged after CLP, renal blood flow (RBF) was decreased 61% by 6 h. Renal microcirculatory analysis showed the number of continuously flowing cortical capillaries decreased significantly from 69 to 48% by 6 h with a 66% decrease in red blood cell velocity and a 57% decline in volumetric flow. The progression of renal microcirculatory hypoperfusion was associated with peritubular capillary leakage and reactive nitrogen species generation. Sham adults had higher mean arterial pressure (118 vs. 69 mmHg), RBF (4.2 vs. 1.1 ml·min(-1)·g(-1)), and peritubular capillary velocity (78% continuous flowing capillaries vs. 69%) compared with pups. CLP produced a greater decrease in renal microcirculation in pups, supporting the notion that adult models may not be the most appropriate for studying pediatric sepsis-induced AKI. Lower RBF and reduced peritubular capillary perfusion in the pup suggest the pediatric kidney may be more susceptible to AKI than would be predicted using adults models.

Intracellular Ca2+ silences L-type Ca2+ channels in mesenteric veins: mechanism of venous smooth muscle resistance to calcium channel blockers.

RATIONALE: Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that Ca(2+) influx through L-type Ca(2+) channels (LTCC) mediates arterial but not venous tone. OBJECTIVE: The object of this study was to resolve the mechanism of venous resistance to CCBs. METHODS AND RESULTS: We compared the sensitivity of depolarization (KCl)-induced constriction of rat small mesenteric arteries (MAs) and veins (MVs) to the dilator effect of CCBs. Initial findings confirmed that nifedipine progressively dilated depolarization-induced constrictions in MAs but not MVs. However, Western blots showed a similar expression of the alpha(1C) pore-forming subunit of the LTCC in both vessels. Patch-clamp studies revealed a similar density of whole-cell Ca(2+) channel current between single smooth muscle cells (SMCs) of MAs and MVs. Based on these findings, we hypothesized that LTCCs are expressed but "silenced" by intracellular Ca(2+) in venous SMCs. After depletion of intracellular Ca(2+) stores by the SERCA pump inhibitor thapsigargin, depolarization-induced constrictions in MVs were blocked 80% by nifedipine suggesting restoration of Ca(2+) influx through LTCCs. Similarly, KCl-induced constrictions were sensitive to block by nifedipine after depletion of intracellular Ca(2+) stores by caffeine, ryanodine, or 2-aminoethoxydiphenyl borate. Cell-attached patch recordings of unitary LTCC currents confirmed rare channel openings during depolarization of venous compared to arterial SMCs, but chelating intracellular Ca(2+) significantly increased the open-state probability of venous LTCCs. CONCLUSIONS: We report that intracellular Ca(2+) inactivates LTCCs in venous SMCs to confer venous resistance to CCB-induced dilation, a fundamental drug property that was previously unexplained.