Rat Cerebral Arteries Research Articles

Intracellular calcium stores contribute to the myogenic response of rat cerebral arteries

Cerebral blood flow is controlled by autoregulatory mechanisms which maintain blood flow within narrow limits despite large changes in blood pressure. Increases in blood pressure cause small cerebral arteries to constrict, a mechanism called the myogenic response. We investigated the contribution of intracellular calcium stores to the development of myogenic tone in rat middle cerebral arteries.Cerebral arteries mounted in a pressure myograph were subjected to stepwise increases in intra‐vascular pressure (20–120 mmHg), and changes in vessel diameter were recorded in the presence or absence of drugs. Cyclopiazonic (CPA; 10 μM), an inhibitor of the SR Ca2+‐ATPase, attenuated myogenic tone at 80mmHg by 64.2 ± 7.2% (n=5). Caffeine (10 mM), a ryanodine channel opener, reduced myogenic tone by 80.3 ± 6.7% (n=5). The effect of caffeine was not inhibited by iberiotoxin (IBTX 100nM; n=4), a large conductance calcium‐activated potassium channel (BKCa) blocker, suggesting that activation of BKCa is unlikely. When CPA was added in combination with nifedipine (10 μM), an L‐type calcium channel blocker, development of myogenic tone was abolished. In conclusion, our data suggests that release of intracellular calcium from the SR plays an important role in the development of myogenic tone in rat middle cerebral arteries. Funded by Heart and Stroke Foundation and University of Alberta Queen Elizabeth II Award.

The Role of IP3 Receptors in Generating Calcium Waves and Cerebral Myogenic Tone

This study of the cerebral vasculature examined whether: 1) elevated intravascular pressure induces asynchronous Ca2+ waves by activating IP3 receptors; and 2) these events drive myogenic tone development by augmenting myosin light chain phosphorylation (LC20). Rat cerebral arteries pressurized between 20–100 mmHg were examined in the absence and presence of agents that deplete the sarcoplasmic reticulum (SR) of Ca2+ or block IP3 receptors. Diameter and membrane potential (VM) were assessed using conventional techniques whereas Ca2+ waves and LC20 phosphorylation were monitored using confocal microscopy and western blot analysis, respectively. Elevated intravascular pressure increased the proportion of smooth muscle cells firing Ca2+ waves as well as the frequency of these voltage‐independent events. Agents that deplete the SR of Ca2+ (ryanodine, CPA and Thapsigargin) or block IP3 receptors (2‐APB or Xestospongin‐C) eliminated Ca2+ wave generation. The loss of Ca2+ waves attenuated LC20 phosphorylation and attenuated myogenic tone development. It did not, however, alter pressure‐induced depolarization in an intact artery In summary, this study shows that pressure‐induced Ca2+ waves facilitate cerebral myogenic tone by providing a proportion of the Ca2+ required to activate myosin light chain kinase. Supported by NSERC.

Glycated proteins inhibit K channels in isolated vascular smooth muscle cells

Fibronectin (FN) has been shown to enhance K+ channel activity via an integrin‐mediated mechanism. As vascular smooth muscle (VSM) K+ channels mediate vasodilation, we investigated whether advanced glycation of fibronectin (as occurs in diabetes and renal failure) alters the normal stimulatory effect of this matrix protein on these channels. Under sterile conditions, FN (1mg/ml) was glycated (gFN) for 5 days in the presence of methylglyoxal (50mM) or glycolaldehyde (50mM). Albumin, a non‐matrix protein, was similarly glycated as a control. VSM cells were enzymatically isolated from rat cerebral arteries for K+ channel activity studies via whole cell patch clamp. The inhibitors, iberiotoxin (0.1μM) and 4‐aminopyridine (1mM), were used to identify contributions of large conductance, Ca2+‐activated, K+ channels and voltage‐gated K+ channels, respectively. While native FN enhanced whole cell K+ current (1.8 fold), gFN caused a 56% inhibition of current compared to baseline. Furthermore, native albumin did not enhance basal K+ current but when glycated caused inhibition (61%; p < 0.05). Inhibitor studies indicated a predominant effect of gFN on the Kv component of total K+ current. These studies provide a potential mechanism by which advanced glycated proteins impair VSM function and adversely impact arteriolar vasodilation.

Association Between Changes in Weight and Cerebral Arteries in Rats

The objective of the study was to gain a better understanding of brain artery diameters and anatomical variations for precise modification of cerebral blood supply in ischemic stroke models. Sprague-Dawley rats (n = 35) were used for the experiment. Rats were perfused and resin replicas of cerebral arteries were created using a corrosion casting technique. Resin replicas were measured and analyzed for correlation of vessel lumen with animal sex and weight. A strong correlation between root of aorta diameter and weight was observed (p < 0.0001). We also observed a significant correlation between weight, internal carotid arteries, right external carotid artery, and pterygopalatine arteries. For the common carotid artery, a significant difference between the left and right branches was observed even though there was no association with weight. There was no significant association observed between animal sex and vessel size independent of weight. A better knowledge of vessel lumen in relation to animal sex and weight is essential for adequate blockage of an intracranial artery to induce cerebral ischemia in a rat model of stroke. This study provides a viable reference for choice of rat size in relation to the size of embolic agents such as filaments, microwires, or in vitro thrombus used in ischemic stroke experiments.

Daidzein relaxes rat cerebral basilar artery via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells

Daidzein, a phytoestrogen, has been reported to produce vasodilation via inhibition of Ca(2+) inflow. However, the involvement of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the effect of daidzein is debated. Therefore, the present study was designed to investigate the effect of daidzein on the rat cerebral basilar artery and the underlying molecular mechanisms. Isolated cerebral basilar artery rings and single vascular smooth muscle cells (VSMCs) were used for vascular reactivity and electrophysiology measurements, to investigate the effect of daidzein on BK(Ca) channels in cerebral basilar artery smooth muscle. In addition, the human BK(Ca) channel alpha-subunit gene (hslo) was transfected into HEK293 cells, to directly assess whether daidzein activates BK(Ca) channels. The results showed that daidzein produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Paxilline, a selective BK(Ca) channel blocker, significantly inhibited the daidzein-induced vasodilation, whereas NS1619, a selective BK(Ca) channel opener, enhanced the vasodilation. In the whole-cell configuration, daidzein increased noisy oscillation currents in cerebral basilar artery VSMCs in a concentration-dependent manner, and washout of daidzein or blockade of BK(Ca) channels with paxilline fully reversed the increase. However, daidzein did not substantially affect hSlo currents in HEK293 cells when applied to the outside of the cell membrane. In conclusion, these results indicate that the activation of BK(Ca) channels in VSMCs at least partly contributes to the daidzein-induced vasodilation of the rat cerebral basilar artery. The beta1-subunit of BK(Ca) channels plays a critical role in the activation of BK(Ca) currents by daidzein.

Molecular Cloning and Expression of the Ryanodine Receptor Type 2 (RyR2) from Rat Cerebral Artery Smooth Muscle

Ryanodine receptors (RyRs) are a family of Ca2+ release channels found in intracellular Ca2+ storage/release organelles and participate in a variety of important Ca2+ signaling phenomena. Here, we set to clone and sequence full-length cDNA encoding the type 2 RyR (RyR2) from smooth muscle cells of Rattus norvegicus cerebral arteries. Middle cerebral and basilar arteries were isolated and de-endothelized, and total RNA was purified for RT-PCR. RyR2 cDNA was divided in two parts of similar size; 5′ segment: 1-7486 bp, and 3′ segment: 7487-14862 bp. Specific primers were designed to obtain both 5′ and 3′-terminals. Following insertion of both terminals in the mammalian expression vector pCI-neo, we characterized several clones by restriction analysis, and confirmed the full-length cDNA sequence by automated sequencing. The rat cerebral artery smooth muscle RyR2 cDNA contains 14862 bp and encodes a deduced protein of 4953 amino acids with a Mr of 562451.3 Da. Nucleotide blast analysis indicates that the cerebral artery smooth muscle RyR2 shows 100% identity with recombinant RyR2 cloned from rat cardiac muscle (Accession NM_032078). RyR2 cDNA expression was determined after transfection of HEK293 cells with the insert into the pCMV6-AN-tagGFP vector. Membrane insertion of cerebral artery myocyte RyR2 was determined by immunolabeling with polyclonal antibodies. To our knowledge, this is the first time that a RyR is cloned from rat cerebral artery smooth muscle, its functional characteristics being currently studied. Supported by Grant AA11560 (AMD).

Strict Structural Requirements for Cholesterol to Inhibit BK Channels Point to Specific Steroid-Protein Interactions

Cholesterol decreases large conductance, voltage/calcium-gated potassium (BK) channel activity (NPo), an action that solely requires the channel pore-forming (α) subunit and a minimum phospholipid environment (Crowley et al., 2003). This cholesterol action is attributed to cholesterol-induced tight packing of bilayer lipids (Chang et al., 1995). Cholesterol modulation of ion channels via direct protein-steroid interactions, however, is increasingly recognized (Epshtein et al., 2009). Cholesterol analogs have been widely used to distinguish between lipid bilayer-mediated and specific protein recognition mechanisms. Thus, we probed cholesterol analogs on BK α subunits cloned from rat cerebral artery myocytes (“cbv1”; AY330293) after channel reconstitution into 3:1 (w/w) POPE:POPS bilayers. Cholesterol (33 mol%) decreased cbv1 NPo by ≈25%. In contrast, 5-cholenic acid-3β-ol, having a carboxyl group at the lateral chain end, failed to decrease NPo, underscoring the importance of a hydrophobic chain for sterol insertion into the bilayer hydrophobic core and channel inhibition. Coprostanol and cholestanol having the A/B junction in cis and trans, respectively, also decreased NPo (≤25%). In contrast, cholesterol, coprostanol and cholestanol epimers, having the C3-hydroxyl group in α-configuration, failed to decrease NPo. Therefore, a β-conformation in the hydroxyl is necessary for these monohydroxy-sterols to inhibit BK channels, strongly suggesting specific, steroid-protein interactions. Moreover, we probed the cbv1 channel with enantiomeric cholesterol (ent-cholesterol), which has physico-chemical properties similar to those of cholesterol yet can be differentially sensed by protein sites, as demonstrated by the lack of viability of C. Elegans when only ent-cholesterol is present (Crowder et al., 2001). Remarkably, ent-cholesterol repeatedly failed to reduce cbv1 NPo, buttressing the idea that cholesterol inhibition of BK channels requires steroid recognition by protein site(s), likely present in the cbv1 subunit itself.Support: AA11560 and HL077424 (AMD), UTHSC Neuroscience Postdoctoral Fellowship (AB).

Subarachnoid hemorrhage induces enhanced expression of thromboxane A 2 receptors in rat cerebral arteries

Cerebral ischemia remains the key cause of morbidity and mortality after subarachnoid hemorrhage (SAH) with a pathogenesis that is still poorly understood. The aim of the present study was to examine the involvement of thromboxane A 2 receptors (TP) in the pathophysiology of cerebral ischemia after SAH in cerebral arteries. SAH was induced in rats by injecting 250 μl of blood into the prechiasmatic cistern. Two days after the SAH, cerebral arteries were harvested and contractile responses to the TP receptor agonist U46619 were investigated with myographs. In addition, the contractile responses were examined after pretreatment with selective TP receptor antagonist GR3219b. The TP receptor RNA and protein levels were analyzed by quantitative real-time PCR and immunohistochemistry, respectively. The global and regional cerebral blood flows (CBFs) were quantified with an autoradiographic technique. SAH resulted in enhanced contractile responses to U46619 as compared to sham. The TP receptor antagonist GR3219b abolished the enhanced contractile responses to U46619 observed after SAH. The TP receptor mRNA level was elevated after SAH as compared to sham. The level of TP receptor protein on the smooth muscle cells (SMCs) was increased in SAH compared to sham. Global and regional CBFs were reduced in SAH as compared to sham. The results demonstrate that SAH results in CBF reduction and this is associated with the enhanced expression of TP receptors in the SMC of cerebral arteries and microvessels.

Involvement of calcium-calmodulin-dependent protein kinase II in endothelin receptor expression in rat cerebral arteries

Experimental cerebral ischemia and organ culture of cerebral arteries result in the enhanced expression of endothelin ET(B) receptors in smooth muscle cells via increased transcription. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CAMK) in the transcriptional expression of endothelin receptors after organ culture. Rat basilar arteries were incubated for 24 h with or without the CAMK inhibitor KN93 or ERK1/2 inhibitor U0126. The contractile responses to endothelin-1 (ET-1; ET(A) and ET(B) receptor agonist) and sarafotoxin 6c (S6c; ET(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of the ET(A) and ET(B) receptors and CAMKII were determined by real-time PCR, and their protein levels were evaluated by immunohistochemistry and Western blot. The mRNA levels of CAMKII and the ET(B) receptor increased during organ culture, but there was no change in the expression of the ET(A) receptor. This effect was abolished by coincubation with KN93 or U0126. In functional studies, both inhibitors attenuated the S6c-induced contraction. Incubating the arteries with KN93, but not U0126, decreased the amount of phosphorylated CAMKII. The inhibitors had no effect on the levels of myosin light chain during organ culture, as measured by Western blot. CAMKII is involved in the upregulation of the endothelin ET(B) receptor and interacts with the ERK1/2 pathway to enhance receptor expression. CAMKII has no effect on the contractile apparatus in rat cerebral arteries.

Channel β2–4 subunits fail to substitute for β1 in sensitizing BK channels to lithocholate

Large conductance, calcium- and voltage-gated potassium (BK) channels regulate numerous physiological processes. While most basic functional characteristics of native BK channels are reproduced by BK α ( slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary β subunits (encoded by KCNMB1–4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate β subunit-containing BK channels, yet these steroids appear to be sensed by different types of β subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that β1 sensitizes the BK channel to μM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits (“cbv1”) with β1, β2, β3 or β4 in Xenopus laevis oocytes we now demonstrate that BK β2, β3 and β4 subunits fail to substitute for β1 in providing LC-sensitivity (150 μM) to the BK channel. These findings document for the first time a rather selective steroid activation of BK channels via a particular channel accessory subunit. In addition, LC routinely activated native BK channels in myocytes freshly isolated from rat cerebral artery smooth muscle, where BK β1 is highly expressed, while failing to do so in skeletal ( flexor digitorum brevis) muscle, where BK β1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by β1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional β1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.

Cerebrovascular Responses to Insulin in Rats

Effects of insulin on cerebral arteries have never been examined. Therefore, we determined cerebrovascular actions of insulin in rats. Both PCR and immunoblot studies identified insulin receptor expression in cerebral arteries and in cultured cerebral microvascular endothelial cells (CMVECs). Diameter measurements (% change) of isolated rat cerebral arteries showed a biphasic dose response to insulin with an initial vasoconstriction at 0.1 ng/mL (-9.7%+/-1.6%), followed by vasodilation at 1 to 100 ng/mL (31.9%+/-1.4%). Insulin also increased cortical blood flow in vivo (30%+/-8% at 120 ng/mL) when applied topically. Removal of reactive oxygen species (ROS) abolished the vasoconstriction to insulin. Endothelial denudation, inhibition of K(+) channels, and nitric oxide (NO) synthase, all diminished insulin-induced vasodilation. Inhibition of cytochrome P450 enhanced vasodilation in endothelium-intact arteries, but promoted vasoconstriction after endothelial denudation. Inhibition of cyclooxygenase abolished vasoconstriction and enhanced vasodilation to insulin in all arteries. Inhibition of endothelin type A receptors enhanced vasodilation, whereas endothelin type B receptor blockade diminished vasodilation. Insulin treatment in vitro increased Akt phosphorylation in cerebral arteries and CMVECs. Fluorescence studies of CMVECs showed that insulin increased intracellular calcium and enhanced the generation of NO and ROS. Thus, cerebrovascular responses to insulin were mediated by complex mechanisms originating in both the endothelium and smooth muscle.

Differential effects of selective PDE5 inhibitors in rat cerebral arteries in vitro and in vivo

Compounds which increase cGMP levels are implicated in migraine pathophysiology as well as stroke recovery. Inhibitors of the cGMP degrading enzyme phosphodiesterase type 5 (PDE5) induce headache and migraine in humans, however surprisingly and unlike other migraine inducing drugs without measurable dilatation of cerebral arteries [1] or changes in hemodynamic response or excitability [2]. We aimed to investigate whether sildenafil and tadalafil induced dilatation of rat middle cerebral and meningeal arteries in vitro and in vivo.

β1 adrenergic receptor‐initiated dilation in rat cerebral arteries is mediated by Shaker‐type KV1 channels and is attenuated in angiotensin II‐induced hypertension

The β1 adrenergic receptor (β1AR), a protein ubiquitously expressed in the cardiovascular system, initiates vasodilation in the cerebral circulation. For example, rat cerebral arteries dilate robustly to isoproterenol (ISO) and norepinephrine (NE), and these responses are blocked by atenolol, a β1AR blocker. However, the identity of the ion channel(s) that mediate the β1AR‐initiated dilation is unclear. In this study, we report that the dilator responses to ISO and NE of isolated, cannulated rat cerebral arteries perfused at 60 mm Hg are abolished by 1 μM correolide, a selective blocker of Shaker‐type voltage‐gated (KV1) channels. To examine the effect of hypertension on this dilation, we used Sprague‐Dawley rats subcutaneously infused with angiotensin II (Ang II, 500 ng/kg/min) for two weeks. Cerebral arteries from Ang II hypertensive rats dilated significantly less to ISO and NE compared to the saline‐infused control rats, suggesting a loss of β1AR‐initiated dilation. Real‐time PCR revealed a reduction in gene expression of pore‐forming α1.2 and α1.5 subunits of KV1 channels in cerebral arteries of Ang II hypertensive rats. Collectively, our study provides evidence for: i) a functional coupling of KV1 channels and β1AR in rat cerebral arteries, and ii) a coordinated loss of this vasodilator pathway in the Ang II rat model of hypertension that may result in cerebral blood flow abnormalities. AHA 0715570Z (BKJ)

Lipid-soluble cigarette smoking particles induce expression of inflammatory and extracellular-matrix-related genes in rat cerebral arteries

Cigarette smoking is one of the strongest risk factors for stroke. However, the underlying molecular mechanisms that smoke leads to the pathogenesis of stroke are incompletely understood. Dimethyl sulfoxide (DMSO)-soluble (lipid-soluble) cigarette smoking particles (DSP) were extracted from cigarette smoke (0.8 mg nicotine per cigarette; Marlboro). Rat cerebral arteries were isolated and organ cultured in the presence of DSP (0.2 microl/ml, equivalent to the plasma level in smokers) for 24 h. The expression of matrix metalloproteinase 9 and 13 (MMP9 and MMP13), angiotensin receptor 1 and 2 (AT(1) and AT(2)), interleukin 6 and inducible nitric oxide synthase (iNOS) were investigated at mRNA level by real-time PCR and/or at protein level by immunohistochemistry. In addition, the activity of three mitogen-activated protein kinases (p38, ERK 1/2 and SAPK/JNK) and their downstream transcription factors (ATF-2, Elk-1 and c-Jun) were examined. We observed that compared with control (DMSO-treated cerebral arteries), the cerebral arteries treated by DSP exhibited enhanced expression of MMP13 and AT(1) receptors, but not of AT(2) receptors, at both mRNA and protein levels, suggesting that a transcriptional mechanism is most likely involved in the DSP effects. This is further supported by the findings that DSP induced phosphorylation of p38 mitogen-activated protein kinases inflammatory signal protein in parallel with activation of its downstream transcription factor ATF-2 and Elk-1. However, ERK 1/2 and SAPK/JNK activities were markedly expressed in the control (organ culture per se with DMSO), and DSP failed to further enhance the activation of ERK 1/2 and SAPK/JNK in the cerebral arteries. DSP induces cerebral vessel inflammation with activation of p38 MAPK inflammatory signal and the downstream transcriptional factors (ATF-2 and Elk-1) in parallel with enhanced extracellular-matrix-related gene transcription and increased AT(1) receptor expression in the cerebral arteries, which are key events in stroke pathogenesis.

Rho‐Kinase‐Mediated Suppression of KDR Current in Cerebral Arteries Requires an Intact Actin Cytoskeleton

This study examined the role of the actin cytoskeleton in Rho‐kinase‐mediated suppression of the delayed rectifier K+ (KDR) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated and whole cell KDR currents monitored using conventional patch clamp electrophysiology. At +40 mV, the KDR current averaged 19.8 ± 1.6 pA/pF (mean ± SE) and was potently inhibited by uridine triphosphate (UTP; 30 µM). Consistent with past observations, KDR current suppression was blocked by the Rho‐kinase inhibitors H‐1152 (300 nM) and Y‐27632 (30 µM). Likewise, disruption of the actin cytoskeleton using cytochalasin D (10 µM) or latrunculin A (10 nM) abolished the ability of UTP to suppress KDR. In intact cerebral arteries, UTP induced actin polymerization in a Rho‐kinase‐dependent manner, suggesting that Rho‐mediated actin reorganization could play a role in the modulation of KDR current. Using pressure myography techniques, UTP‐induced depolarization and constriction of cerebral arteries were found to be significantly reduced following Rho‐kinase inhibition with H‐1152, and following the disruption of the actin cytoskeleton using cytochalasin D or latrunculin A. We conclude from our electrophysiological, biochemical, and functional observations that Rho‐kinase mediated alteration of the actin cytoskeleton likely underlies the inhibition of KDR current by UTP, ultimately contributing to the depolarization and constriction of cerebral arteries.

Electrophysiological Characterization of a TRPC‐like Current in Cerebral Arterial Smooth Muscle

The goal of this investigation was to isolate and characterize a TRPC‐like current in cerebral arterial smooth muscle cells (VSMCs). Briefly, VSMCs were enzymatically isolated from rat cerebral arteries and whole cell currents monitored using patch clamp electrophysiology. Our initial experiments revealed two subpopulations of VSMCs that displayed either an outwardly rectifying or a doubly rectifying current. The reversal potential of both currents shifted with the Na(+) equilibrium potential and were potently blocked by micromolar concentrations of Gd(3+). A pharmacological characterization subsequently revealed that the doubly rectifying TRPC‐like current was insensitive to Cl(‐) channels inhibitors (DIDS and niflumic acid), amiloride and flufemamate. In contrast, tamoxifen elicited a dual effect both attenuating and activating this current at submicromolar and micromolar concentrations, respectively. Similar to findings from cultured smooth muscle cells, hyposmotic challenge and vasoconstrictor agonists (UTP) activated this TRPC‐like non selective cation current. To summarize, this study is the first to characterize the electrical and pharmacological properties of a doubly‐rectifying TRPC‐like cation current in cerebral VSMCs. Ongoing investigations continue to define the molecular composition of this TRPC‐like current along with it's mechanisms of physiological regulation.

High voltage activated, nifedipine‐sensitive and insensitive calcium channels are present in rat cerebral arteries

Whilst L‐type voltage dependent calcium channels (VDCCs) are reported to mediate cerebral vasoconstriction, selective antagonists are ineffective in treating vasospasm. We therefore investigated the expression and characteristics of VDCC subtypes in adult rat cerebral vessels. Quantitative PCR revealed expression of seven subtypes, with the T‐type, CaV3.1, and L‐type, CaV1.2, channels the most highly expressed. Immunohistochemistry confirmed protein expression for both channels. Isolated smooth muscle cells (holding potential ‐70 or ‐100mV) displayed inward currents that were activated at ‐40mV and maximal at 0‐10mV (10mM BaCl2 as charge carrier). The current at +10mV had an activation constant (tact) of 1.92 ±0.02ms and two inactivation constants (tinact) of 68.9±15.5ms and &gt;300ms. No additional current was found when cells were held at ‐100mV. Nifedipine (1μΜ reduced the peak current in a non‐time dependent fashion, revealing a nifedipine‐insensitive component, that activated and inactivated significantly faster (tact= 1.11±0.06ms; tinact= 23.5±5.6ms). and was abolished by the further addition of the putative T‐type channel blocker mibefradil. When applied singly, either mibefradil (1μM) or NNC‐55 0396 (1μM) abolished all evoked current. We conclude that a nifedipine‐insensitive VDCC with high voltage activation contributes to calcium influx in cerebral arteries in addition to CaV1.2.

The CaV1.2 α1 subunit N‐terminus regulates cerebral artery contractility

Voltage‐dependent Ca2+ (Cav1.2) channels are the major Ca2+ entry pathway in arterial myocytes and are essential for myogenic constriction. Although Cav1.2 α1 subunits undergo splicing, physiological functions of vascular splice variants are unclear. We have shown that arterial myocytes express two different Cav1.2 α1 subunit N‐terminal variants (J Biol Chem, 282: 29211‐21, 2007); a novel isoform from exon 1c (e1c) and the previously described exon 1b (e1b). Here, we examined protein expression and physiological functions of Cav1.2 α1 subunit N‐termini in resistance‐size rat cerebral arteries. Custom antibodies recognizing peptide sequences encoded by e1b and e1c detected Cav1.2 α1 subunits in cerebral arteries. ShRNA targeting e1b and e1c both reduced Cav1.2 α1 subunit expression, with e1c shRNA causing a larger reduction than did e1b shRNA. E1b or e1c overexpression reduced pressure (60 mmHg)‐induced myogenic tone, with e1c causing a greater reduction than did e1b. Acute application of a membrane‐permeant peptide corresponding to the sequence encoded by e1c inhibited myocyte voltage‐dependent Ba2+ currents and reversibly dilated pressurized arteries. In contrast, a control peptide had no effect. In summary, data suggest that Cav1.2 α1 subunits containing e1b and e1c are expressed in cerebral artery myocytes and that e1c regulates voltage‐dependent Ca2+ currents and myogenic tone.

Cerebrovascular actions of insulin

Insulin resistance is a major risk factor for cerebrovascular and neurodegenerative diseases; however, cerebrovascular actions of insulin have not been studied. Therefore, we evaluated the vascular actions of insulin in isolated rat cerebral arteries (CAs). Diameter measurements of CAs showed a biphasic response to insulin; an initial constriction was followed by vasodilation. Endothelial denudation or large conductance calcium‐activated K+ channels (KCa) inhibition resulted in vasoconstriction to insulin. Inhibition of small/intermediate conductance KCa or inward‐rectifier or voltage gated or ATP dependent K+ channels reduced insulin induced vasodilation. Inhibition of nitric oxide (NO) synthase or cytochrome P450 reduced vasodilation while inhibition of cyclooxygenase enhanced vasodilation to insulin. Inhibition of endothelin type A receptors enhanced the vasodilation while type B blockade diminished vasodilation to insulin. Inhibition of reactive oxygen species (ROS) production or scavenging the ROS abolished the vasoconstriction to insulin. Fluorescence studies of cultured rat cerebral microvascular endothelial cells showed elevation of intracellular calcium and enhanced generation of NO and ROS in response to insulin. Thus, insulin induced vasodilation was dependent K+ channels and endothelial factors while vasoconstriction was mediated by ROS, prostanoids and endothelin.

Inhibition of voltage-gated L-type calcium channels by labedipinedilol-A involves protein kinase C in rat cerebrovascular smooth muscle cells

Labedipinedilol-A, a novel calcium channel blocker with α/β-adrenoceptor blockade properties, inhibits L-type calcium channels (LTCCs) in rat cerebrovascular smooth muscle cells (CSMCs). We used conventional whole cell patch-clamp electrophysiology to investigate Ba 2+ currents ( I Ba) through LTCCs in rat CSMCs enzymatically dissociated from rat cerebral arteries. Labedipinedilol-A (1, 10 µM) reversibly inhibited I Ba in a voltage-dependent manner without modifying the I Ba current–voltage relationship. The I Ba was also abolished by the LTCC blocker nifedipine (1 µM), but enhanced by the LTCC activator Bay K8644 (100 nM). Labedipinedilol-A shifted the steady-state inactivation curve of I Ba to more negative potentials. Additionally, labedipinedilol-A had greater inhibitory activity on I Ba holding at − 40 mV than at − 80 mV. This might contribute to labedipinedilol-A's more selective effect on vascular muscles compared to cardiac muscles. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and norepinephrine-enhanced I Ba were also inhibited by labedipinedilol-A. Pretreatment with the PKC inhibitor chelerythrine (5 µM) attenuated labedipinedilol-A-mediated I Ba inhibition. However, the Rho kinase inhibitor Y-27632 (30 µM) had little effect on labedipinedilol-A inhibition of I Ba. Labedipinedilol-A inhibition of voltage-dependent LTCCs may be, at least in part, due to its modulation of the PKC pathway.