Subarachnoid hemorrhage (SAH) following cerebral aneurysm rupture is associated with substantial morbidity and mortality. The ability of SAH to induce vasospasm in large diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is unclear. Conversely, little is known regarding the impact of SAH on intracerebral (parenchymal) arterioles, which are critical for regulation of cerebral blood flow. To assess the function of parenchymal arterioles following SAH, measurements of diameter, intracellular Ca2+ ([Ca2+]i) and membrane potential were performed in intact arterioles from unoperated (control), sham-operated and SAH model rats. At physiological intravascular pressure, parenchymal arterioles from SAH animals exhibited significantly elevated [Ca2+]i and enhanced constriction compared with arterioles from control and sham-operated animals. Elevated [Ca2+]i and enhanced tone following SAH were observed in the absence of vascular endothelium and were abolished by the L-type voltage-dependent Ca2+ channel (VDCC) inhibitor nimodipine. Molecular assessment of the L-type VDCC CaV1.2 indicated unchanged mRNA and protein expression in arterioles from SAH animals. Increased CaV1.2 activity following SAH may also reflect enhanced pressure-induced membrane potential depolarization of arteriolar smooth muscle. Membrane potential measurements in arteriolar myocytes using intracellular microelectrodes revealed approximately 7 mV depolarization at 40 mmHg in myocytes from SAH animals. Further, when membrane potential was adjusted to similar values, arteriolar [Ca2+]i and tone were similar between groups. These results demonstrate that greater pressure-dependent membrane potential depolarization results in increased activity of CaV1.2 channels, elevated [Ca2+]i and enhanced constriction of parenchymal arterioles from SAH animals. Thus, impaired regulation of parenchymal arteriolar [Ca2+]i and diameter may restrict cerebral blood flow in SAH patients. Although nimodipine is used clinically to prevent delayed neurological deficits in SAH patients, the use of this drug has been limited by hypotension and treatment options remain inadequate. Therefore, our next objective was to explore strategies to selectively suppress CaV1.2 channels in the cerebral vasculature. To do so, we examined the physiological role of smooth muscle CaV1.2 splice variants containing the alternatively-spliced exon 9* in cerebral artery constriction. Using antisense oligonucleotides, we demonstrate that suppression of exon 9*-containing CaV1.2 splice variants results in substantially reduced cerebral artery constriction to elevated extracellular [K+]. In addition, no further reduction in constriction was observed following suppression of all Cav1.2 splice variants, suggesting that exon 9* splice variants are functionally dominant in cerebral artery constriction. In summary, results shown in this dissertation demonstrate that increased CaV1.2 activity following SAH results in enhanced constriction of parenchymal arterioles. Furthermore, evidence is provided supporting the concept that CaV1.2 splice variants with exon 9* are critical for cerebral artery constriction and may provide a novel target for the prevention of delayed ischemic deficits in SAH patients.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-10-28T14:21:00Z No. of bitstreams: 2 Dissertação - Carolina de Fátima Reis - 2015.pdf: 1195189 bytes, checksum: e77613f54e17ab204bf8a94706552ef6 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-28T14:47:30Z (GMT) No. of bitstreams: 2 Dissertação - Carolina de Fátima Reis - 2015.pdf: 1195189 bytes, checksum: e77613f54e17ab204bf8a94706552ef6 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-28T14:47:30Z (GMT). No. of bitstreams: 2 Dissertação - Carolina de Fátima Reis - 2015.pdf: 1195189 bytes, checksum: e77613f54e17ab204bf8a94706552ef6 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-06-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The use of medicinal plants for the treatment of hypertension treatment has been extensively studied. The genus Pterodon spp. Vogel (Fabaceae), popularly known as sucupira, has five native Brazilian cerrado species. Studies demonstrated that the Pterodon spp has antispasmodic and myorelaxant activities. Phytochemical studies of sucupira found furanic diterpenes with vouacapanic skeleton in their fruits, and the therapeutics activities from this gender are essentially attributed to these compounds. The aim of this study was to evaluete the possible vasorelaxant activity and the mechanisms of action of the oil-resin of sucupira (SOR) and its isolated diterpene, methyl-6α-acetoxy-7β-hydroxyvouacapan-17β-oate in isolated rat aorta preparations. For comparison, verapamil curves (1 nM – 100 μM) were also obtained. The results demonstrated that both, S0R (0 – 56 μg/mL) and the isolated diterpene (0 – 48 μg/mL) have a reversible concentration-dependent vasorrelaxant activity. Endothelium denudation did not impair in the relaxant effect of both, ORS and the diterpene. Aortic rings KCl-stimulated obtained a lower IC50 value than rings contracted with phenylephrine under increasing concentrations of the tested substances. The SOR and diterpene relaxant activity was not impaired when nonselective K+ channels blockers were used (Tetraethylammonium). The use of cyclopiazonic acid (SERCA inhibitor) indicated that there was no involvement of the Ca2+ reuptake by sarcoplasmatic reticulum and the use of ODQ (an inhibitor of guanylyl cyclase) and MDL-12,330A (an inhibitor of adenylyl cyclase), showed that the 3, 5- cyclic guanosine monophosphate (cGMP) and 3',5'-cyclic adenosine monophosphate (cAMP) pathways were not involved in SOR relaxant activity. However, the results indicate a possibly activity of the soluble guanylyl ciclase on diterpene relaxant effect. Both, SOR and isolated diterpene inhibited the CaCl2 induced contractions in aortic rings stimulated with phenylephrine (0.1μM) or Bay K8644 (a Cav1.2 channel activator; 1μM) and by depolarizing KCl solution (40 mM). Computational molecular docking studies demonstrated that the vasodilator effect of diterpene relies on blocking the Cav1.2 channel, and patch clamp results showed that diterpene substantially decreased the ionic current through Cav1.2 in freshly dissociated vascular smooth muscle cells. These findings suggest that SOR and its isolated diterpene induce endothelium-independent vascular relaxation by blocking the L-type Ca2+ channel (Cav1.2). / O uso de plantas medicinais para o tratamento da hipertensão tem sido bastante estudado. O gênero Pterodon spp. Vogel (Fabaceae), popularmente conhecido como sucupira, possui cinco espécies nativas do Cerrado brasileiro. Estudos indicam que Pterodon spp possui efeito antiespasmódico e miorrelaxante. A caracterização fitoquímica da sucupira constatou a presença de diterpenos com esqueleto furânico do tipo vouacapânico em seus frutos, sendo esses metabólitos provavelmente responsáveis pelas funções terapêuticas do gênero. O objetivo deste estudo foi avaliar a possível atividade vasorrelaxante bem como os possíveis mecanismos de ação do oleorresina de sucupira (ORS) e do seu diterpeno isolado, 6α-acetoxi-7β-hidroxivouacapano-17β-oato de metila, em preparações isoladas de aorta de ratos. Para fins de comparação, curvas para verapamil (1 nM – 100 μM) foram obtidas. Os resultados indicaram que tanto o ORS (0 – 56 μg/mL) quanto o diterpeno (0 – 48 μg/mL) possuem atividade vasorrelaxante reversível de maneira concentração-dependente. A remoção do endotélio vascular não alterou a atividade tanto do ORS quanto do seu diterpeno. Preparações estimuladas por KCl obtiveram um menor valor de IC50 que preparações previamente contraídas por fenilefrina quando submetidas a concentrações crescentes das substâncias teste. O relaxamento induzido pelo ORS e diterpeno não foi alterado na presença do inibidor não seletivo de canal de K+, tetraetilamônio. Não houve envolvimento quanto à mobilização intracelular de Ca2+ quando as preparações foram incubadas com inibidor da SERCA, o ácido cliclopiazônico. A utilização de ODQ, inibidor da guanilato ciclase solúvel e MDL-12,330A, inibidor da adenilato ciclase, demonstrou que as vias dos nucleotídeos cíclicos guanosina 3, 5- monofosfato cíclico (GMPc) e adenosina 3',5'-monofosfato cíclico (AMPc), também não estão envolvidas na atividade do ORS, embora suponha-se possível envolvimento da guanilato ciclase solúvel sGC na atividade vasorrelaxante do diterpeno. Ambos, ORS e diterpeno, impediram a contração induzida por solução de CaCl2 em preparações estimuladas por fenilefrina (0,1μM) ou Bay K8644 (ativador específico de Cav1.2; 1μM) e por estímulo despolarizante com solução de KCl (40 mM). Estudos de docking molecular demonstraram que o efeito vasodilatador do diterpeno baseia-se no bloqueio de Cav1.2 e os resultados obtidos pela técnica de patch-clamp com o diterpeno mostraram substancial diminuição da corrente iónica através Cav1.2 em células de músculo liso vascular recentemente dissociados. Estes resultados sugerem que ORS e seu diterpeno isolado induzem relaxamento vascular endotélio independente, bloqueando o canal de Ca2+ do tipo L (Cav1.2).

Voltage-gated Ca2+ (Cav) channels are activated upon depolarization. They specifically allow Ca2+ ions to come into the cell. These Ca2+ ions are bi-functional because they not only control cell excitability but also couple electrical activity to complex downstream signaling events, such as excitation-contraction coupling in muscles and neurotransmitter release in neurons. In the brain, Cav channels are expressed in the pre- or post-synaptic membrane of most excitable cells, neurons. In the past few years, their expression and function have also been characterized in many nonexcitable cells such as astrocytes. This dissertation focuses on the regulation of one subtype of postsynaptic Cav channels, Cav1.2, in neurons. In the first part of chapter I, I provide a literature overview of Cav channels in terms of their subtypes, localizations, physiological functions, and biophysical properties. For years, Cav channels were studied as single entities. But now, based on multiple proteomic studies, we know that these channels actually do not live alone. They interact with numerous proteins depending on the physiological conditions. Such interactions can anchor the channels to optimal sites of action, and tether Cav channels to their modulatory molecules. Therefore, it is crucial to understand how Cav channels are regulated by their macromolecular assembly. Among these protein partners, our lab studied the regulation of Cav channels by a subset of PDZ-domain containing proteins. Because these proteins play an important role in scaffolding and they colocalize with both pre- and post-synaptic Cav channels. Indeed, previous studies from our lab and other groups have revealed that PDZ proteins participate in a multitude of Cav regulation. The second part of chapter I introduces the diverse modulation of neuronal Cav channels by numerous PDZ proteins. In neurons, Cav1.2 channels regulate neuronal excitability and synaptic plasticity. Their functions have been implicated in learning, memory, and mood regulation. A study published in the journal Lancet showed that the gene encoding Cav1.2 is a common risk factor for five major psychiatric disorders. A PDZ protein, densin-180 (densin) is an excitatory synapse protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin exhibit similar behavioral phenotypes that closely match those in mice lacking Cav1.2. In chapter II and III, we investigated the functional impact of densin on Cav1.2 channels and their auxiliary subunit β2a. Besides the regulation of Cav channels by their interactome, we have also known for a long time that Ca2+ currents undergo a negative feedback regulation. This regulation is called Ca2+-dependent inactivation (CDI) and it is mediated by Ca2+ that directly traverses the pore. CDI has been described for Cav channels in multiple cell types. In the heart, CDI prevents excessively long cardiac action potentials, which in turn can prevent activity-dependent arrhythmia. In neurons, CDI may be neuroprotective by preventing excitotoxic Ca2+ overloads. In the last 18 years, two essential components have been revealed in the mechanism of CDI. One is the protein calmodulin (CaM). CaM interacts directly with sites on the C-terminus of Cav channels. It binds to the incoming Ca2+ ions and produces a mysterious conformational change that determines the conductance of the channel. The other molecular player is Cavβ protein family. Cavβ comprises four subfamilies β1 through β4, which generally enhance the channel inactivation, except β2a. In chapter IV, Xiaohan Wang from Roger Colbran’s lab in Vanderbilt University, and I identified a new molecular determinant for Cav1.2 CDI. The α2δ subunit is an extracellular component of the Cav channel complex. Similar to Cavβ subunits, α2δ subunits are essential for the biophysical properties, surface level, and trafficking of Cavα1 subunits. There are four isoforms of α2δ subunits (α2δ1 to α2δ4). They display distinct tissue distributions. Although the roles of α2δ subunits in Cav channel regulation were studied extensively, studies have proposed that the function of α2δ subunits may be in part or entirely independent of Cav channel complex, such as synaptogenesis. Considering the important role of α2δ in physiology and pathology, it is imperative to identify the factors that regulate the properties of α2δ. In chapter V, I explored the trafficking dynamics of α2δ1 and revealed a potential regulator of α2δ1 for its protein stability and localization. One beauty of doing research is that it always motivates us to think and ask more questions on our journey of demystifying nature. While looking at the evidence that I find, I realize how much more we could do in the future. In chapter VI, I conclude the findings of each chapter and share my perspectives on the future direction for these research projects.

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