Sung W. Rhee

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.

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.

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.

The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10.

Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER‐resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best‐practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous β1,4‐galactosyltransferase and UDP‐N‐acetylgalactosamine:polypeptide N‐acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady‐state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.

The β3 Subunit Contributes to Vascular Calcium Channel Upregulation and Hypertension in Angiotensin II–Infused C57BL/6 Mice

Voltage-gated L-type Ca2+ (Cav1.2) channels in vascular smooth muscle cells are a predominant Ca2+ influx pathway that mediates arterial tone. Channel biogenesis is accomplished when the pore-forming &agr;1C subunit coassembles with regulatory Cav&bgr; subunits intracellularly, and the multiprotein Cav1.2 channel complex translocates to the plasma membrane to form functional Ca2+ channels. We hypothesized that the main Cav&bgr; isoform in vascular smooth muscle cells, Cav&bgr;3, is required for the upregulation of arterial Cav1.2 channels during the development of hypertension, an event associated with abnormal Ca2+-dependent tone. Cav1.2 channel expression and function were compared between second-order mesenteric arteries of C57BL/6 wild-type (WT) and Cav&bgr;3−/− 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 Cav1.2 channel expression and accentuated Ca2+-mediated contractions compared with saline-infused WT mice. In contrast, Cav1.2 channels failed to upregulate in mesenteric arteries of Ang II–infused Cav&bgr;3−/− mice, and Ca2+-dependent reactivity was normal in these arteries. Basal systolic blood pressure was not significantly different between WT and Cav&bgr;3−/− mice (98 ± 2 and 102 ± 3 mm Hg, respectively), but the Cav&bgr;3−/− mice showed a blunted pressor response to Ang II infusion. Two weeks after the start of Ang II administration, the systolic blood pressure of Cav&bgr;3−/− mice averaged 149 ± 4 mm Hg compared with 180 ± 5 mm Hg in WT mice. Thus, the Cav&bgr;3 subunit is a critical regulatory protein required to upregulate arterial Cav1.2 channels and fully develop Ang II-dependent hypertension in C57BL/6 mice.

Loss of cerebrovascular Shaker-type K+ channels: a shared vasodilator defect of genetic and renal hypertensive rats

The cerebral arteries of hypertensive rats are depolarized and highly myogenic, suggesting a loss of K(+) channels in the vascular smooth muscle cells (VSMCs). The present study evaluated whether the dilator function of the prominent Shaker-type voltage-gated K(+) (K(V)1) channels is attenuated in middle cerebral arteries from two rat models of hypertension. Block of K(V)1 channels by correolide (1 micromol/l) or psora-4 (100 nmol/l) reduced the resting diameter of pressurized (80 mmHg) cerebral arteries from normotensive rats by an average of 28 +/- 3% or 26 +/- 3%, respectively. In contrast, arteries from spontaneously hypertensive rats (SHR) and aortic-banded (Ao-B) rats with chronic hypertension showed enhanced Ca(2+)-dependent tone and failed to significantly constrict to correolide or psora-4, implying a loss of K(V)1 channel-mediated vasodilation. Patch-clamp studies in the VSMCs of SHR confirmed that the peak K(+) current density attributed to K(V)1 channels averaged only 5.47 +/- 1.03 pA/pF, compared with 9.58 +/- 0.82 pA/pF in VSMCs of control Wistar-Kyoto rats. Subsequently, Western blots revealed a 49 +/- 7% to 66 +/- 7% loss of the pore-forming alpha(1.2)- and alpha(1.5)-subunits that compose K(V)1 channels in cerebral arteries of SHR and Ao-B rats compared with control animals. In each case, the deficiency of K(V)1 channels was associated with reduced mRNA levels encoding either or both alpha-subunits. Collectively, these findings demonstrate that a deficit of alpha(1.2)- and alpha(1.5)-subunits results in a reduced contribution of K(V)1 channels to the resting diameters of cerebral arteries from two rat models of hypertension that originate from different etiologies.

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 α 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.Rationale: Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that Ca2+ influx through L-type Ca2+ 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 &agr;1C pore-forming subunit of the LTCC in both vessels. Patch-clamp studies revealed a similar density of whole-cell Ca2+ 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 Ca2+ in venous SMCs. After depletion of intracellular Ca2+ stores by the SERCA pump inhibitor thapsigargin, depolarization-induced constrictions in MVs were blocked 80% by nifedipine suggesting restoration of Ca2+ influx through LTCCs. Similarly, KCl-induced constrictions were sensitive to block by nifedipine after depletion of intracellular Ca2+ 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 Ca2+ significantly increased the open-state probability of venous LTCCs. Conclusions: We report that intracellular Ca2+ inactivates LTCCs in venous SMCs to confer venous resistance to CCB-induced dilation, a fundamental drug property that was previously unexplained.

Vascular Smooth Muscle-Specific Knockdown of the Noncardiac Form of the L-Type Calcium Channel by MicroRNA-Based Short Hairpin RNA as a Potential Antihypertensive Therapy

In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (CaL) current and vascular tone is increased because of increased expression of the noncardiac form of the CaL (Cav1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Cav1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotransfected with a rat Cav1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific enhancer/promoter reduced endogenous Cav1.2 expression by 61% and decreased the CaL current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Cav1.2, it did not affect the CaL expression and function in cultured cardiomyocytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Cav1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Cav1.2 siRNA without similarly affecting cardiac CaL expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension.

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

Non‐Technical Summary  Shaker‐type potassium channels are found on the smooth muscle cells of blood vessels in the brain and are important in keeping the blood vessels open or dilated. We show that a protein called PSD95, previously found in nerve cells, interacts with these potassium channels. PSD95 may act as a scaffold to ensure that the potassium channels are expressed in adequate numbers and in the right location on the smooth muscle cells. When we reduced the number of PSD95 proteins, we saw that the potassium channels were also reduced and the blood vessels were not as dilated compared to blood vessels with normal amounts of PSD95. This research may help us understand how abnormal constriction of blood vessels in the brain occurs in diseases such as high blood pressure and stroke.

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 KV1.2&agr; 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 KV1.2&agr; 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.