Quantal Ca2+ release mediated by very few IP3 receptors that rapidly inactivate allows graded responses to IP3.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that link extracellular stimuli to Ca2+ signals. Ca2+ release from intracellular stores is "quantal": low IP3 concentrations rapidly release a fraction of the stores. Ca2+ release then slows or terminates without compromising responses to further IP3 additions. The mechanisms are unresolved. Here, we synthesize a high-affinity partial agonist of IP3Rs and use it to demonstrate that quantal responses do not require heterogenous Ca2+ stores. IP3Rs respond incrementally to IP3 and close after the initial response to low IP3 concentrations. Comparing functional responses with IP3 binding shows that only a tiny fraction of a cell's IP3Rs mediate incremental Ca2+ release; inactivation does not therefore affect most IP3Rs. We conclude, and test by simulations, that Ca2+ signals evoked by IP3 pulses arise from rapid activation and then inactivation of very few IP3Rs. This allows IP3Rs to behave as increment detectors mediating graded Ca2+ release.

Selexipague para pacientes adultos com hipertensão arterial pulmonar (HAP ­ Grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta / Selexipag for adult patients with pulmonary arterial hypertension (PAH ­ Group I) in functional class III who have not achieved a satisfactory response with ERA and/or PDE5i, as an alternative to iloprost

CONTEXTO: A Hipertensão Arterial Pulmonar (HAP) é uma doença rara, grave, caracterizada por disfunção endotelial e remodelamento das artérias pulmonares, pertencente a um grupo de distúrbios conhecidos como hipertensão pulmonar (HP). A incidência anual da HAP varia entre 1,9 a 3,7 novos casos/milhão de habitantes, enquanto a prevalência estimada é de 10,6 a 16 casos/milhão de habitantes. A HAP é uma condição progressiva com prognóstico ruim, que pode levar rapidamente à incapacidade e morte prematura. Devido a sua baixa incidência é uma doença classificada como doença rara. Para o tratamento da HAP utiliza-se associação de terapias específicas e a combinação de medicamentos com diferentes mecanismos de ação. O PCDT de HAP comtempla para tratamento 5 medicamentos aprovados pela ANVISA, que incluem antagonistas do receptor de endotelina (ERA) (ambrisentana e bosentana); inibidores da fosfodiesterase 5 (PDE5i) (sildenafila) e atuantes na via da prostaciclina (PGI2), que são os análogos da PGI2 (iloprosta). O iloprosta é um medicamento administrado por via inalatória de 6 a 9 vezes por dia. Selexipague é um agonista seletivo do receptor IP, administrado por via oral, duas vezes ao dia, proporcionando ao paciente maior facilidade de administração e comodidade posológica indicado para o tratamento de longo prazo da HAP. A indicação de incorporação é tratamento com selexipague para pacientes adultos com hipertensão arterial pulmonar (HAP ­ Grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta. TECNOLOGIA: Selexipague (Uptravi®). PERGUNTA: O uso de selexipague é eficaz, seguro e custo-efetivo no tratamento de pacientes com HAP em pacientes com hipertensão arterial pulmonar quando comparado às terapias atualmente disponíveis no SUS? EVIDÊNCIAS CIENTÍFICAS: Foi realizada uma revisão sistemática da literatura com o objetivo de identificar todas as evidências disponíveis que avaliassem a eficácia e segurança de selexipague em comparação com as terapias atualmente disponíveis no SUS em pacientes adultos com HAP. No total, dez estudos foram incluídos pelo demandante (1 ECR, com 4 análises post-hoc e 5 revisões sistemáticas com meta-análise). Na avaliação crítica foi excluída uma metanálise totalizando 9 estudos. A evidência principal de selexipague é o ECR Fase III GRIPHON, em que pacientes foram randomizados para receber selexipague ou placebo em adição a terapia de base (80% já usavam ERA e/ou PDE5i). O risco do desfecho primário composto de morte, progressão da doença ou de uma complicação relacionada a HAP foi significativamente menor entre os pacientes que receberam selexipague do que entre os que receberam placebo (41,6% dos pacientes no grupo placebo e 27,0% no grupo selexipague [HR no grupo selexipague em comparação ao grupo placebo: 0,60; IC99% 0,46-0,78; p 0,60; p<0,001. Em relação aos eventos adversos 41 patients (7.1%) no grupo placebo e 82 pacientes (14.3%) no grupo selexipague descontinuaram o regime do estudo por evento adverso. Os principais eventos para o selexipague foram dor de cabeça (em 3.3% dos pacientes), diarreia (em 2.3%), e náusea (em 1.7%). Na semana 26, a proporção de pacientes sem piora ou melhora na classe functional entre os grupos selexipague e placebo não foram significativas (74.9% and 77.8%, respectivamente; OR, 1.16; 99% IC, 0.81 to 1.66; P=0.28). As metanálises incluídas foram de análises indiretas entre o selexipague e iloprosta com resultados muito frágeis devido principalmente a grande heterogeneidade de participantes nos estudos, regimes de tratamento e desfechos. AVALIAÇÃO ECONÔMICA: Foi elaborado um modelo de custo-utilidade pelo demandante onde foi utilizada uma abordagem simples e direta para comparar selexipague com iloprosta em pacientes com HAP grupo I e classe funcional III. A análise econômica conduzida baseou-se em um modelo de Markov para projetar a relação entre custo e anos de vida ajustados pela qualidade (AVAQ) entre selexipague comparado a Iloprosta. Foi utilizado como desfecho clínico a diferença em utilidade, considerando a via de administração dos medicamentos (oral vs inalatória). O resultado da razão de custo efetividade incremental (RCEI) do caso base, considerando a terapia combinada e o cenário onerado, foi de R$ 320.721,97. AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO: A incorporação do selexipague ao SUS, geraria um impacto incremental de R$ 16,6 milhões no primeiro ano de incorporação, totalizando incremento de R$ 67,9 milhões em 5 anos se a taxa de difusão for até 70%. Caso fosse considerado o cenário de desoneração do selexipague, o IO em 5 anos seria de R$ 11,9 milhões. Em um cenário mais agressivo elaborado internamente chegando a uma difusão de 95% em 5 anos o impacto incremental chegaria a R$ 72,5 milhões. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: Detectaram-se três tecnologias para compor o esquema terapêutico de adultos com adultos com HAP, Grupo I e CF-III, que não alcançaram resposta satisfatória com ERA e/ou PDE5-i, ralinepague, riociguate e sotatercepte. Sendo que este último não possui registro nas Agências regulatórias consultadas. CONSIDERAÇÕES FINAIS: Não há na literatura evidências diretas que comparem selexipague com iloprosta em monoterapia ou terapia combinada com ERA e/ou PDE5i. O estudo GRIPHON não apresentou análise completa de subgrupos de pacientes em terapia combinada e por classe funcional que mostre a real eficácia nos pacientes classe funcional III. As evidências apresentadas demonstram que os resultados clínicos de selexipague em relação ao placebo reduzem a morbimortalidade da doença. Essa evidência em relação ao iloprosta fica inferida indiretamente como equivalente por serem da mesma classe de medicamentos. RECOMENDAÇÃO PRELIMINAR: Diante do exposto, a Conitec, em sua 97ª reunião ordinária, realizada no dia 06 de maio de 2021, deliberou que a matéria fosse disponibilizada em Consulta Pública com recomendação preliminar de todos os membros do plenário desfavorável à incorporação de selexipague para tratamento de pacientes adultos com Hipertensão Arterial Pulmonar (HAP -grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta no SUS. CONSULTA PÚBLICA: A Consulta Pública nº 51 foi realizada entre os dias 07/06/2021 e 28/06/2021. Foram recebidas 359 contribuições, sendo 72 pelo formulário para contribuições técnico-científicas e 287 pelo formulário para contribuições sobre experiência ou opinião de pacientes, familiares, amigos ou cuidadores de pacientes, profissionais de saúde ou pessoas interessadas no tema. A grande maioria das contribuições não concordou com a não incorporação e apenas 3 contribuições foram concordantes. Ao final, o Plenário da Conitec entendeu que houve nova proposta de preço do medicamento além do entendimento da comodidade posológica do medicamento em relação ao iloprosta o que fez com que a plenária mudasse seu entendimento sobre o tema, fazendo com que sua recomendação preliminar passasse a ser favorável à incorporação do Selexipague. DELIBERAÇÃO FINAL: Pelo exposto, o Plenário da Conitec, em sua 99ª Reunião Ordinária, no dia 01 de julho de 2021, deliberou por unanimidade recomendar a incorporação do Selexipague para pacientes adultos com Hipertensão arterial pulmonar (HAP ­ grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i como alternativa a iloprosta. A incorporação favorável está condicionada ao Protocolo Clínico e Diretrizes Terapêuticas do Ministério da Saúde. Assim, foi assinado o Registro de Deliberação nº 637/2021. DECISÃO: Incorporar o selexipague para pacientes adultos com hipertensão arterial pulmonar (HAP - Grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta, no âmbito do Sistema Único de Saúde ­ SUS, conforme a Portaria nº 53, publicada no Diário Oficial da União nº 149, seção 1, páginas 60 e 61, em 9 de agosto de 2021.

Selexipague para pacientes adultos com hipertensão arterial pulmonar (HAP ­ Grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta / Selexipag for adult patients with pulmonary arterial hypertension (PAH - Group I) in functional class III who did not achieve a satisfactory response with ERA and/or PDE5i, such as alternative to iloprost

CONTEXTO: A Hipertensão Arterial Pulmonar (HAP) é uma doença rara, grave, caracterizada por disfunção endotelial e remodelamento das artérias pulmonares, pertencente a um grupo de distúrbios conhecidos como hipertensão pulmonar (HP). A incidência anual da HAP varia entre 1,9 a 3,7 novos casos/milhão de habitantes, enquanto a prevalência estimada é de 10,6 a 16 casos/milhão de habitantes. A HAP é uma condição progressiva com prognóstico ruim, que pode levar rapidamente à incapacidade e morte prematura. Devido a sua baixa incidência é uma doença classificada como doença rara. Para o tratamento da HAP utiliza-se associação de terapias específicas e a combinação de medicamentos com diferentes mecanismos de ação. O PCDT de HAP comtempla para tratamento 5 medicamentos aprovados pela ANVISA, que incluem antagonistas do receptor de endotelina (ERA) (ambrisentana e bosentana); inibidores da fosfodiesterase 5 (PDE5i) (sildenafila) e atuantes na via da prostaciclina (PGI2), que são os análogos da PGI2 (iloprosta). O iloprosta é um medicamento administrado por via inalatória de 6 a 9 vezes por dia. Selexipague é um agonista seletivo do receptor IP, administrado por via oral, duas vezes ao dia, proporcionando ao paciente maior facilidade de administração e comodidade posológica indicado para o tratamento de longo prazo da HAP. A indicação de incorporação é tratamento com selexipague para pacientes adultos com hipertensão arterial pulmonar (HAP ­ Grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta. Tecnologia: Selexipague (Uptravi®). PERGUNTA: O uso de selexipague é eficaz, seguro e custo-efetivo no tratamento de pacientes com HAP em pacientes com hipertensão arterial pulmonar quando comparado às terapias atualmente disponíveis no SUS? EVIDÊNCIAS CIENTÍFICAS: Foi realizada uma revisão sistemática da literatura com o objetivo de identificar todas as evidências disponíveis que avaliassem a eficácia e segurança de selexipague em comparação com as terapias atualmente disponíveis no SUS em pacientes adultos com HAP. No total, dez estudos foram incluídos pelo demandante (1 ECR, com 4 análises post-hoc e 5 revisões sistemáticas com meta-análise). Na avaliação crítica foi excluída uma metanálise totalizando 9 estudos. A evidência principal de selexipague é o ECR Fase III GRIPHON, em que pacientes foram randomizados para receber selexipague ou placebo em adição a terapia de base (80% já usavam ERA e/ou PDE5i). O risco do desfecho primário composto de morte, progressão da doença ou de uma complicação relacionada a HAP foi significativamente menor entre os pacientes que receberam selexipague do que entre os que receberam placebo (41,6% dos pacientes no grupo placebo e 27,0% no grupo selexipague [HR no grupo selexipague em comparação ao grupo placebo: 0,60; IC99% 0,46-0,78; p 0,60; p<0,001]). Em relação aos eventos adversos 41 patients (7.1%) no grupo placebo e 82 pacientes (14.3%) no grupo selexipague descontinuaram o regime do estudo por evento adverso. Os principais eventos para o selexipague foram dor de cabeça (em 3.3% dos pacientes), diarreia (em 2.3%), e náusea (em 1.7%). Na semana 26, a proporção de pacientes sem piora ou melhora na classe functional entre os grupos selexipague e placebo não foram significativas (74.9% and 77.8%, respectivamente; OR, 1.16; 99% IC, 0.81 to 1.66; P=0.28). As metanálises incluídas foram de análises indiretas entre o selexipague e iloprosta com resultados muito frágeis devido principalmente a grande heterogeneidade de participantes nos estudos, regimes de tratamento e desfechos. AVALIAÇÃO ECONÔMICA: Foi elaborado um modelo de custo-utilidade pelo demandante onde foi utilizada uma abordagem simples e direta para comparar selexipague com iloprosta em pacientes com HAP grupo I e classe funcional III. A análise econômica conduzida baseou-se em um modelo de Markov para projetar a relação entre custo e anos de vida ajustados pela qualidade (AVAQ) entre selexipague comparado a Iloprosta. Foi utilizado como desfecho clínico a diferença em utilidade, considerando a via de administração dos medicamentos (oral vs inalatória). O resultado da razão de custo efetividade incremental (RCEI) do caso base, considerando a terapia combinada e o cenário onerado, foi de R$ 320.721,97. AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO: A incorporação do selexipague ao SUS, geraria um impacto incremental de R$ 16,6 milhões no primeiro ano de incorporação, totalizando incremento de R$ 67,9 milhões em 5 anos se a taxa de difusão for até 70%. Caso fosse considerado o cenário de desoneração do selexipague, o IO em 5 anos seria de R$ 11,9 milhões. Em um cenário mais agressivo elaborado internamente chegando a uma difusão de 95% em 5 anos o impacto incremental chegaria a R$ 72,5 milhões. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: Detectaram-se três tecnologias para compor o esquema terapêutico de adultos com adultos com HAP, Grupo I e CF-III, que não alcançaram resposta satisfatória com ERA e/ou PDE5-i, ralinepague, riociguate e sotatercepte. Sendo que este último não possui registro nas Agências regulatórias consultadas. CONSIDERAÇÕES FINAIS: Não há na literatura evidências diretas que comparem selexipague com iloprosta em monoterapia ou terapia combinada com ERA e/ou PDE5i. O estudo GRIPHON não apresentou análise completa de subgrupos de pacientes em terapia combinada e por classe funcional que mostre a real eficácia nos pacientes classe funcional III. As evidências apresentadas demonstram que os resultados clínicos de selexipague em relação ao placebo reduzem a morbimortalidade da doença. Essa evidência em relação ao iloprosta fica inferida indiretamente como equivalente por serem da mesma classe de medicamentos. RECOMENDAÇÃO PRELIMINAR: Diante do exposto, a Conitec, em sua 97ª reunião ordinária, realizada no dia 06 de maio de 2021, deliberou que a matéria fosse disponibilizada em Consulta Pública com recomendação preliminar de todos os membros do plenário desfavorável à incorporação de selexipague para tratamento de pacientes adultos com Hipertensão Arterial Pulmonar (HAP -grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta no SUS. CONSULTA PÚBLICA: A Consulta Pública nº 51 foi realizada entre os dias 07/06/2021 e 28/06/2021. Foram recebidas 359 contribuições, sendo 72 pelo formulário para contribuições técnico-científicas e 287 pelo formulário para contribuições sobre experiência ou opinião de pacientes, familiares, amigos ou cuidadores de pacientes, profissionais de saúde ou pessoas interessadas no tema. A grande maioria das contribuições não concordou com a não incorporação e apenas 3 contribuições foram concordantes. Ao final, o Plenário da Conitec entendeu que houve nova proposta de preço do medicamento além do entendimento da comodidade posológica do medicamento em relação ao iloprosta o que fez com que a plenária mudasse seu entendimento sobre o tema, fazendo com que sua recomendação preliminar passasse a ser favorável à incorporação do Selexipague. DELIBERAÇÃO FINAL: Pelo exposto, o Plenário da Conitec, em sua 99ª Reunião Ordinária, no dia 01 de julho de 2021, deliberou por unanimidade recomendar a incorporação do Selexipague para pacientes adultos com Hipertensão arterial pulmonar (HAP ­ grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i como alternativa a iloprosta. A incorporação favorável está condicionada ao Protocolo Clínico e Diretrizes Terapêuticas do Ministério da Saúde. Assim, foi assinado o Registro de Deliberação nº 637/2021. DECISÃO: Incorporar o selexipague para pacientes adultos com hipertensão arterial pulmonar (HAP - Grupo I) em classe funcional III que não alcançaram resposta satisfatória com ERA e/ou PDE5i, como alternativa a iloprosta, no âmbito do Sistema Único de Saúde ­ SUS, conforme a Portaria nº 53, publicada no Diário Oficial da União nº 149, seção 1, páginas 60 e 61, em 9 de agosto de 2021.

Both d- and l-Glucose Polyphosphates Mimic d-myo-Inositol 1,4,5-Trisphosphate: New Synthetic Agonists and Partial Agonists at the Ins(1,4,5)P3 Receptor.

Chiral sugar derivatives are potential cyclitol surrogates of the Ca2+-mobilizing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Six novel polyphosphorylated analogues derived from both d- and l-glucose were synthesized. Binding to Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R] and the ability to release Ca2+ from intracellular stores via type 1 Ins(1,4,5)P3Rs were investigated. ß-d-Glucopyranosyl 1,3,4-tris-phosphate, with similar phosphate regiochemistry and stereochemistry to Ins(1,4,5)P3, and α-d-glucopyranosyl 1,3,4-tris-phosphate are full agonists, being equipotent and 23-fold less potent than Ins(1,4,5)P3, respectively, in Ca2+-release assays and similar to Ins(1,4,5)P3 and 15-fold weaker in binding assays. They can be viewed as truncated analogues of adenophostin A and refine understanding of structure-activity relationships for this Ins(1,4,5)P3R agonist. l-Glucose-derived ligands, methyl α-l-glucopyranoside 2,3,6-trisphosphate and methyl α-l-glucopyranoside 2,4,6-trisphosphate, are also active, while their corresponding d-enantiomers, methyl α-d-glucopyranoside 2,3,6-trisphosphate and methyl α-d-glucopyranoside 2,4,6-trisphosphate, are inactive. Interestingly, both l-glucose-derived ligands are partial agonists: they are among the least efficacious agonists of Ins(1,4,5)P3R yet identified, providing new leads for antagonist development.

d-chiro-Inositol Ribophostin: A Highly Potent Agonist of d-myo-Inositol 1,4,5-Trisphosphate Receptors: Synthesis and Biological Activities.

Analogues of the Ca2+-releasing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [1, Ins(1,4,5)P3] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by d-chiro-inositol in d-chiro-inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P3 mimic ribophostin 6 may increase the activity. d-chiro-Inositol ribophostin 10 was synthesized by coupling as building blocks suitably protected ribose 12 with l-(+)-3-O-trifluoromethylsulfonyl-6-O-p-methoxybenzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 11. Separable diastereoisomeric 3-O-camphanate esters of (±)-6-O-p-methoxy-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol allowed the preparation of 11. Selective trans-isopropylidene deprotection in coupled 13, then monobenzylation gave separable regioisomers 15 and 16. p-Methoxybenzyl group deprotection of 16, phosphitylation/oxidation, then deprotection afforded 10, which was a full agonist in Ca2+-release assays; its potency and binding affinity for Ins(1,4,5)P3R were similar to those of adenophostin. Both 4 and 10 elicited a store-operated Ca2+ current ICRAC in patch-clamped cells, unlike Ins(1,4,5)P3 consistent with resistance to metabolism. d-chiro-Inositol ribophostin is the most potent small-molecule Ins(1,4,5)P3 receptor agonist without a nucleobase yet synthesized.

Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

Cryo-EM reveals ligand induced allostery underlying InsP3R channel gating.

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are cation channels that mobilize Ca2+ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP3R activation is the coupled interplay between binding of InsP3 and Ca2+ that switches the ion conduction pathway between closed and open states to enable the passage of Ca2+ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here, we present the electron cryo-microscopy structure of InsP3R1 from rat cerebellum determined to 4.1 Å resolution in the presence of activating concentrations of Ca2+ and adenophostin A (AdA), a structural mimetic of InsP3 and the most potent known agonist of the channel. Comparison with the 3.9 Å-resolution structure of InsP3R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP3R channel.

Region-specific proteolysis differentially regulates type 1 inositol 1,4,5-trisphosphate receptor activity.

The inositol 1,4,5 trisphosphate receptor (IP3R) is an intracellular Ca2+ release channel expressed predominately on the membranes of the endoplasmic reticulum. IP3R1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood. Our previous work has demonstrated that IP3R1 channels, formed following either enzymatic fragmentation or expression of the corresponding complementary polypeptide chains, retain tetrameric architecture and are still activated by IP3 binding despite the loss of peptide continuity. In this study, we demonstrate that region-specific receptor fragmentation modifies channel regulation. Specifically, the agonist-evoked temporal Ca2+ release profile and protein kinase A modulation of Ca2+ release are markedly altered. Moreover, we also demonstrate that activation of fragmented IP3R1 can result in a distinct functional outcome. Our work suggests that proteolysis of IP3R1 may represent a novel form of modulation of IP3R1 channel function and increases the repertoire of Ca2+ signals achievable through this channel.

Changes in IP3 Receptor Expression and Function in Aortic Smooth Muscle of Atherosclerotic Mice.

Peroxynitrite is an endothelium-independent vasodilator that induces relaxation via membrane hyperpolarization. The activation of IP3 receptors triggers the opening of potassium channels and hyperpolarization. Previously we found that relaxation to peroxynitrite was maintained during the development of atherosclerosis due to changes in the expression of calcium-regulatory proteins. In this study we investigated: (1) the mechanism of peroxynitrite-induced relaxation in the mouse aorta, (2) the effect of atherosclerosis on relaxation to peroxynitrite and other vasodilators, and (3) the effect of atherosclerosis on the expression and function of the IP3 receptor. Aortic function was studied using wire myography, and atherosclerosis was induced by fat-feeding ApoE-/- mice. The expression of IP3 receptors was studied using Western blotting and immunohistochemistry. Relaxation to peroxynitrite was attenuated by the IP3 antagonists 2-APB and xestospongin C and also the Kv channel blocker 4-aminopyridine (4-AP). Atherosclerosis attenuated vasodilation to cromakalim and the AMPK activator A769662 but not peroxynitrite. Relaxation was attenuated to a greater extent by 2-APB in atherosclerotic aortae despite the reduced expression of IP3 receptors. 4-AP was less effective in ApoE-/- mice fat-fed for 4 months. Peroxynitrite relaxation involves an IP3-induced calcium release and KV channel activation. This mechanism becomes less important as atherosclerosis develops, and relaxation to peroxynitrite may be maintained by increased calcium extrusion.

ß2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

Beta adrenergic receptors (ßARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied ßAR, ß2AR, whose ligands are used for asthma and cardiovascular disease. ßARs signal through Gαs G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of ßAR signaling and its disruption in disease. Using fluorescence-based Ca2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby ß2AR activation leads to robust Ca2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP3) receptors. This pathway did not involve cAMP, Gαs, or Gαi or the participation of the other members of the canonical ß2AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca2+ mobilization by ß2AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of ßAR-directed drugs.

At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries.

AIMS: Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca2+/purinergic signalling in controlling blood vessel contraction. We hypothesized that hemichannel Ca2+ entry and ATP release contributes to smooth muscle cell (SMC) Ca2+ dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca2+ and ATP signalling to investigate their influence on SMC Ca2+ dynamics and vessel contractility. METHODS AND RESULTS: Confocal Ca2+ imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca2+ oscillations were inhibited by blocking IP3 receptors with xestospongin-C and by interfering with hemichannel function, most notably by the specific Cx43 hemichannel blocking peptide TAT-L2 and by TAT-CT9 that promotes Cx43 hemichannel opening. Evidence for hemichannel involvement in SMC function was supported by the fact that TAT-CT9 significantly increased SMC resting cytoplasmic Ca2+ concentration, indicating it facilitated Ca2+ entry, and by the observation that norepinephrine-triggered vessel ATP release was blocked by TAT-L2. Myograph tension measurements on isolated SMAs showed significant inhibition of norepinephrine-triggered contractility by the ATP receptor antagonist suramin, but the strongest effect was observed with TAT-L2 that gave ∼80% inhibition at 37 °C. TAT-L2 inhibition of vessel contraction was significantly reduced in conditional Cx43 knockout animals, indicating the effect was Cx43 hemichannel-dependent. Computational modelling suggested these results could be explained by the opening of a single hemichannel per SMC. CONCLUSIONS: These results indicate that Cx43 hemichannels contribute to SMC Ca2+ dynamics and contractility, by facilitating Ca2+ entry, ATP release, and purinergic signalling.

Inositol 1,4,5-trisphosphate-mediated sarcoplasmic reticulum-mitochondrial crosstalk influences adenosine triphosphate production via mitochondrial Ca2+ uptake through the mitochondrial ryanodine receptor in cardiac myocytes.

AIMS: Elevated levels of inositol 1,4,5-trisphosphate (IP3) in adult cardiac myocytes are typically associated with the development of cardiac hypertrophy, arrhythmias, and heart failure. IP3 enhances intracellular Ca(2+ )release via IP3 receptors (IP3Rs) located at the sarcoplasmic reticulum (SR). We aimed to determine whether IP3-induced Ca(2+ )release affects mitochondrial function and determine the underlying mechanisms. METHODS AND RESULTS: We compared the effects of IP3Rs- and ryanodine receptors (RyRs)-mediated cytosolic Ca(2+ )elevation achieved by endothelin-1 (ET-1) and isoproterenol (ISO) stimulation, respectively, on mitochondrial Ca(2+ )uptake and adenosine triphosphate (ATP) generation. Both ET-1 and isoproterenol induced an increase in mitochondrial Ca(2+ )(Ca(2 +) m) but only ET-1 led to an increase in ATP concentration. ET-1-induced effects were prevented by cell treatment with the IP3 antagonist 2-aminoethoxydiphenyl borate and absent in myocytes from transgenic mice expressing an IP3 chelating protein (IP3 sponge). Furthermore, ET-1-induced mitochondrial Ca(2+) uptake was insensitive to the mitochondrial Ca(2+ )uniporter inhibitor Ru360, however was attenuated by RyRs type 1 inhibitor dantrolene. Using real-time polymerase chain reaction, we detected the presence of all three isoforms of IP3Rs and RyRs in murine ventricular myocytes with a dominant presence of type 2 isoform for both receptors. CONCLUSIONS: Stimulation of IP3Rs with ET-1 induces Ca(2+ )release from the SR which is tunnelled to mitochondria via mitochondrial RyR leading to stimulation of mitochondrial ATP production.

Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

New paradigm for pulmonary arterial hypertension treatment.

PURPOSE OF REVIEW: Pulmonary arterial hypertension (PAH) was previously considered a uniformly fatal disease, with patients succumbing to right heart failure and death at an average of 3 years after diagnosis. The past 20 years, however, have seen the development of numerous targeted therapies that have changed the natural history of PAH. As more pharmacologic agents have been approved and utilized, further advances in the design of and endpoints for clinical trials. This study will review some of these notable developments. RECENT FINDINGS: The successful design and completion of long-term, event-driven trials is exemplified in three recent studies: SERAPHIN, GRIPHON, and AMBITION. SERAPHIN and GRIPHON evaluated the newer agents, macitentan, an endothelin receptor antagonist, and selexipag, a prostacyclin receptor agonist, respectively. Both trials were large-scale studies that, in addition to showing marked effect on the primary endpoint of morbidity/mortality, clearly demonstrated that assessment of long-term effects of PAH therapies is feasible for new compounds. The AMBITION study evaluated a treatment strategy, namely up-front combination therapy with tadalafil and ambrisentan compared with monotherapy and showed the combination approach to be superior at decreasing the likelihood of clinical failure. SUMMARY: The evolution of clinical trials in PAH has direct implications for care of these patients. The short and long-term benefits of combination regimens suggest that the multidrug approach to PAH should, in fact, be standard of care for this disease.

Mechanisms of cytotoxicity induced by the anesthetic isoflurane: the role of inositol 1,4,5-trisphosphate receptors.

Isoflurane can induce widespread cytotoxicity. We hypothesized that isoflurane induces apoptosis partly by causing excessive calcium release from the endoplasmic reticulum (ER) via direct activation of inositol 1,4,5-trisphosphate receptors (IP3R). Rat pheochromocytoma cells cultured for seven days with nerve growth factor were divided into four groups: control group (C), IP3R antagonist group (X), isoflurane group (I) and isoflurane + IP3R antagonist group (I+X). Groups I and I+X were treated with 1 MAC isoflurane for 12 h. Groups X and I+X were pretreated with IP3R antagonist. Annexin V/PI apoptosis and TUNEL assays were performed to evaluate cell apoptosis. TEM was used to observe changes in cell ultrastructure. Changes in calcium concentration ([Ca(2+)]i) in the cytoplasm were measured by flow cytometry. RT-PCR was performed to evaluate IP3R mRNA expression. TEM showed that isoflurane treatment altered cell ultrastructure. Compared to group C, cell apoptosis rate and [Ca(2+)]i increased in groups I and I+X (P < 0.05). Compared to group C, IP3R mRNA expression was lower in group X and higher in group I (P < 0.05). Compared to group X, cell apoptosis rate, [Ca(2+)]i and IP3R mRNA expression increased in groups I and I+X (P < 0.05). Compared to group I, cell apoptosis rate, [Ca(2+)]i and IP3R mRNA expression decreased in group I+X (P < 0.05). These results suggest that exposure to 1 MAC isoflurane for 12 h causes excessive calcium release partly by direct activation of IP3R on the ER membrane and triggers cell apoptosis.

Autophagy protects meniscal cells from glucocorticoids-induced apoptosis via inositol trisphosphate receptor signaling.

Intra-articular injection of glucocorticoids (GCs) has been widely used in the management of osteoarthritis and rheumatoid arthritis. Nevertheless, several studies showed that GCs had toxic effects on chondrocytes as well as synovial cells. Previously we reported the protective role of autophagy in the degeneration of meniscal tissues. However, the effects of GCs on autophagy in the meniscal cells have not been fully elucidated. To investigate whether GCs can regulate autophagy in human meniscal cells, the meniscal cells were cultured in vitro and exposed in the presence of dexamethasone. The levels of apoptosis and autophagy were investigated via flow cytometry as well as western blotting analysis. The changes of the aggrecanases were measured using real-time PCR. The role of autophagy in dexamethasone-induced apoptosis was investigated using pharmacological agents and RNA interference technique. An agonist of inositol 1,4,5-trisphosphate receptor (IP3R) was used to investigate the mechanism of dexamethasone-induced autophagy. The results showed that dexamethasone induced autophagy as well as apoptosis in normal human meniscal cells. Using RNA interference technique and pharmacological agents, our results showed that autophagy protected the meniscal cells from dexamethasone-induced apoptosis. Our results also indicated that dexamethasone increased the mRNA levels of aggrecanases. This catabolic effect of dexamethasone was enhanced by 3-MA, the autophagy inhibitor. Furthermore, our results showed that dexamethasone induced autophagy via suppressing the phosphorylation of IP3R. In summary, our results indicated that autophagy protected meniscal cells from GCs-induced apoptosis via inositol trisphosphate receptor signaling.

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells.

KEY POINTS: Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. ABSTRACT: Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.

Triazolophostins: a library of novel and potent agonists of IP3 receptors.

IP3 receptors are channels that mediate the release of Ca(2+) from the intracellular stores of cells stimulated by hormones or neurotransmitters. Adenophostin A (AdA) is the most potent agonist of IP3 receptors, with the ß-anomeric adenine contributing to the increased potency. The potency of AdA and its stability towards the enzymes that degrade IP3 have aroused interest in AdA analogs for biological studies. The complex structure of AdA poses problems that have necessitated optimization of synthetic conditions for each analog. Such lengthy one-at-a-time syntheses limit access to AdA analogs. We have addressed this problem by synthesizing a library of triazole-based AdA analogs, triazolophostins, by employing click chemistry. An advanced intermediate having all the necessary phosphates and a ß-azide at the anomeric position was reacted with various alkynes under Cu(i) catalysis to yield triazoles, which upon deprotection gave triazolophostins. All eleven triazolophostins synthesized are more potent than IP3 and some are equipotent with AdA in functional analyses of IP3 receptors. We show that a triazole ring can replace adenine without compromising the potency of AdA and provide facile routes to novel AdA analogs.

Scaffold hopping with virtual screening from IP3 to a drug-like partial agonist of the inositol trisphosphate receptor.

Inositol 1,4,5-trisphosphate (IP3 ) is a universal signalling molecule that releases calcium from stores within cells by activating the IP3 receptor. Although chemical tools that modulate the IP3 receptor exist, none is ideal due to trade offs between potency, selectivity and cell permeability, and their chemical properties make them challenging starting points for optimisation. Therefore, to find new leads, we used virtual screening to scaffold hop from IP3 by using the program ROCS to perform a 3D ligand-based screen of the ZINC database of purchasable compounds. We then used the program FRED to dock the top-ranking hits into the IP3 binding pocket of the receptor. We tested the 12 highest-scoring hits in a calcium-release bioassay and identified SI-9 as a partial agonist. SI-9 competed with [(3) H]IP3 binding, and reduced histamine-induced calcium signalling in HeLa cells. SI-9 has a novel 2D scaffold that represents a tractable lead for designing improved IP3 receptor modulators.

Systems modeling of Ca(2+) homeostasis and mobilization in platelets mediated by IP3 and store-operated Ca(2+) entry.

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca(2+)]cyt) and high dense tubular system calcium ([Ca(2+)]dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP3) to trigger calcium mobilization from stores and elevation of [Ca(2+)]cyt. Another major source of [Ca(2+)]cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca(2+)]dts drops. A 34-species systems model employed kinetics describing IP3-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca(2+)-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP3-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>-70 mV) and low resting IP3 concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP3 caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets' phenotypes during hemostasis or thrombosis.