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Cerebral hypoperfusion and vascular dysfunction are closely related to common risk factors for ischemic stroke such as hypertension, dyslipidemia, diabetes, and smoking. The role of inhibitory G protein-dependent receptor (GiPCR) signaling in regulating cerebrovascular functions remains largely elusive. We examined the importance of GiPCR signaling in cerebral blood flow (CBF) and its stability after sudden interruption using various in vivo high-resolution magnetic resonance imaging techniques. To this end, we induced a functional knockout of GiPCR signaling in the brain vasculature by injection of pertussis toxin (PTX). Our results show that PTX induced global brain hypoperfusion and microvascular collapse. When PTX-pretreated animals underwent transient unilateral occlusion of one common carotid artery, CBF was disrupted in the ipsilateral hemisphere resulting in the collapse of the cortically penetrating microvessels. In addition, pronounced stroke features in the affected brain regions appeared in both MRI and histological examination. Our findings suggest an impact of cerebrovascular GiPCR signaling in the maintenance of CBF, which may be useful for novel pharmacotherapeutic approaches to prevent and treat cerebrovascular dysfunction and stroke.

Ischemic events are associated with severe inflammation and are here referred to as ischemic inflammatory response (IIR). Recent studies identified the formation of platelet–neutrophil complexes (PNC) as key players in IIR. We investigated the role of extracellular platelet nucleotide signaling in the context of IIR and defined a cybernetic circle, including description of feedback loops. Cybernetic circles seek to integrate different levels of information to understand how biological systems function. Our study specifies the components of the cybernetic system of platelets in IIR and describes the theoretical progression of IIR passing the cybernetic cycle with positive and negative feedback loops based on nucleotide-dependent signaling and functional regulation. The cybernetic components and feedback loops were explored by cytometry, immunohistological staining, functional blocking antibodies, and ADP/ATP measurements. Using several ex vivo and in vivo approaches we confirmed cybernetic parameters, such as controller, sensor, and effector (VASP phosphorylation, P2Y12, ADORAs and GPIIb/IIIa activity), as well as set points (ADP, adenosine) and interfering control and disturbance variables (ischemia). We demonstrate the impact of the regulated platelet–neutrophil complex (PNC) formation in blood and the resulting damage to the affected inflamed tissue. Taken together, extracellular nucleotide signaling, PNC formation, and tissue damage in IIR can be integrated in a controlled cybernetic circle of platelet function, as introduced through this study.

Pertussis toxin (PTX) is a potent virulence factor in patients suffering from whooping cough, but in its detoxified version, it is applied for vaccination. It is thought to contribute to the pathology of the disease including various CNS malfunctions. Based on its enzymatic activity, PTX disrupts GPCR-dependent signaling by modifying the α-subunit of heterotrimeric Gi/o-proteins. It is also extensively used as a research tool to study neuronal functions in vivo and in vitro. However, data demonstrating the penetration of PTX from the blood into the brain are missing. Here, we examined the Gαi/o-modifying activity of PTX in murine brains after its parenteral application. Ex vivo biodistribution analysis of [124I]-PTX displayed poor distribution to the brain while relatively high concentrations were visible in the pancreas. PTX affected CNS and endocrine functions of the pancreas as shown by open-field and glucose tolerance tests, respectively. However, while pancreatic islet Gαi/o-proteins were modified, their neuronal counterparts in brain tissue were resistant towards PTX as indicated by different autoradiographic and immunoblot SDS-PAGE analyses. In contrast, PTX easily modified brain Gαi/o-proteins ex vivo. An attempt to increase BBB permeability by application of hypertonic mannitol did not show PTX activity on neuronal G proteins. Consistent with these findings, in vivo MRI analysis did not point to an increased blood-brain barrier (BBB) permeability following PTX treatment. Our data demonstrate that the CNS is protected from PTX. Thus, we hypothesize that the BBB hinders PTX to penetrate into the CNS and to deliver its enzymatic activity to brain Gαi/o-proteins. KEY MESSAGES: i.p. applied PTX is poorly retained in the brain while reaches high concentration in the pancreas. Pancreatic islet Gαi/o- but not cerebral Gαi/o-proteins are modified by i.p. administered PTX. Gαi/o-proteins from isolated cerebral cell membranes were easily modified by PTX ex vivo. CNS is protected from i.p. administered PTX. PTX does not permeabilize the BBB.

Diabetes mellitus is a chronic, progressive, incompletely understood metabolic disorder whose prevalence has been increasing steadily worldwide. Even though little attention has been paid to lung disorders in the context of diabetes, its prevalence has recently been challenged by newer studies of disease development. In this review, we summarize and discuss the role of diabetes mellitus involved in the progression of pulmonary diseases, with the main focus on pulmonary fibrosis, which represents a chronic and progressive disease with high mortality and limited therapeutic options.

Pertussis toxin (PTX) is a potent virulence factor in patients suffering from whooping cough, but in its detoxified version, it is applied for vaccination. It is thought to contribute to the pathology of the disease including various CNS malfunctions. Based on its enzymatic activity, PTX disrupts GPCR-dependent signaling by modifying the α-subunit of heterotrimeric G

OBJECTIVE The physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis. RESEARCH DESIGN AND METHODS Gene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKIα and Iβ rescue mice [α/βRM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca2+ ([Ca2+]i) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo. RESULTS Immunohistochemistry showed that cGKI is present in α- but not in β-cells in islets of Langerhans. Mice that lack α-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca2+]i levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca2+]i or hormone release in cGKI-deficient islets. CONCLUSIONS We propose that cGKI modulates glucagon release by suppression of [Ca2+]i in α-cells.

It has been suggested that cGMP kinase I (cGKI) dampens cardiac hypertrophy. We have compared the effect of isoproterenol (ISO) and transverse aortic constriction (TAC) on hypertrophy in WT [control (CTR)] mice, total cGKI-KO mice, and cGKIβ rescue mice (βRM) lacking cGKI specifically in cardiomyocytes (CMs). Infusion of ISO did not change the expression of cGKI in the hearts of CTR mice or βRM but raised the heart weight by ∼20% in both. An identical hypertrophic growth response was measured in CMs from CTR mice and βRM and in isolated adult CMs cultured with or without 1 μM ISO. In both genotypes, ISO infusion induced similar changes in the expression of hypertrophy-associated cardiac genes and significant elevation of serum atrial natriuretic peptide and total cardiac cGMP. No differences in cardiac hypertrophy were obtained by 7-day ISO infusion in 4- to 6-week-old conventional cGKI-KO and CTR mice. Furthermore, TAC-induced hypertrophy of CTR mice and βRM was not different and did not result in changes of the cGMP-hydrolyzing phosphodiesterase activities in hypertropic hearts or CMs. These results strongly suggest that cardiac myocyte cGKI does not affect the development of heart hypertrophy induced by pressure overload or chronic ISO infusion.

Aims Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac β1-adrenoceptor overexpression. Methods and results β1-transgenic mice lacking Gαi2 (β1-tg/Gαi2 -/-) were compared with wild-type mice and littermates either overexpressing cardiac β1-adrenoceptors (β1-tg) or lacking Gαi2 (Gαi2 -/-). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or β1-tg, but similar to β1-tg/Gαi2 -/-, mice. Beyond 300 days, mortality of β1-tg/Gαi2 -/- mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, β1-tg, and Gαi2 -/- mice, but significant impairment for β1-tg/Gαi2 -/- mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2 -/- and 394 ± 80% in β1-tg/Gαi2 -/-, respectively). Conclusions Gαi2 deficiency combined with cardiac β1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, β1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased β1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism. Fil: Keller, Kirsten. Universitat Zu Köln; Alemania Fil: Maass, Martina. University Hospital of Cologne; Alemania Fil: Dizayee, Sara. Universitat Zu Köln; Alemania Fil: Leiss, Veronika. Eberhard Karls University Hospitals and Clinics; Alemania Fil: Annala, Suvi. Universitat Zu Köln; Alemania Fil: Köth, Jessica. Universitat Zu Köln; Alemania Fil: Seemann, Wiebke K.. Universitat Zu Köln; Alemania Fil: Müller Ehmsen, Jochen. Asklepios Klinik Altona; Alemania Fil: Mohr, Klaus. Universitaet Bonn; Alemania Fil: Nürnberg, Bernd. Eberhard Karls University Hospitals and Clinics; Alemania Fil: Engelhardt, Stefan. Universitat Technical Zu Munich; Alemania Fil: Herzig, Stefan. Universitat Zu Köln; Alemania Fil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Matthes, Jan. Universitat Zu Köln; Alemania

Insulin and glucagon are the major hormones of the islets of Langerhans that are stored and released from the B- and A-cells, respectively. Both hormones are secreted when the intracellular cytosolic Ca2+ concentration ([Ca2+]i) increases. The [Ca2+]i is modulated by mutual inhibition and activation of different voltage-gated ion channels. The precise interplay of these ion channels in either glucagon or insulin release is unknown, owing in part to the difficulties in distinguishing A- from B-cells in electrophysiological experiments. We have established a single-cell RT-PCR method to identify A- and B-cells from the mouse. A combination of PCR, RT-PCR, electrophysiology and pharmacology enabled us to characterize the different sodium and calcium channels in mouse islet cells. In both A- and B-cells, 60% of the inward calcium current (ICa) is carried by L-type calcium channels. In B-cells, the predominant calcium channel is CaV1.2, whereas CaV1.2 and CaV1.3 were identified in A-cells. These results were confirmed by using mice carrying A- or B-cell-specific inactivation of the CaV1.2 gene. In B-cells, the remaining ICa flows in equal amounts through CaV2.1, CaV2.2 and CaV2.3. In A-cells, 30 and 15% of ICa is due to CaV2.3 and CaV2.1 activity, respectively, whereas CaV2.2 current was not found in these cells. Low-voltage-activated T-type calcium channels could not be identified in A- and B-cells. Instead, two TTX-sensitive sodium currents were found: an early inactivating and a residual current. The residual current was only recovered in a subpopulation of B-cells. A putative genetic background for these currents is NaV1.7.

Bordetella pertussis toxin (PTx), also known as islet-activating protein, induces insulin secretion by ADP-ribosylation of inhibitory G proteins. PTx-induced insulin secretion may result either from inactivation of Gα(o) proteins or from combined inactivation of Gα(o), Gα(i1), Gα(i2), and Gα(i3) isoforms. However, the specific role of Gα(i2) in pancreatic β-cells still remains unknown. In global (Gα(i2)(-/-)) and β-cell-specific (Gα(i2)(βcko)) gene-targeted Gα(i2) mouse models, we studied glucose homeostasis and islet functions. Insulin secretion experiments and intracellular Ca²⁺ measurements were used to characterize Gα(i2) function in vitro. Gα(i2)(-/-) and Gα(i2)(βcko) mice showed an unexpected metabolic phenotype, i.e., significantly lower plasma insulin levels upon intraperitoneal glucose challenge in Gα(i2)(-/-) and Gα(i2)(βcko) mice, whereas plasma glucose concentrations were unchanged in Gα(i2)(-/-) but significantly increased in Gα(i2)(βcko) mice. These findings indicate a novel albeit unexpected role for Gα(i2) in the expression, turnover, and/or release of insulin from islets. Detection of insulin secretion in isolated islets did not show differences in response to high (16 mM) glucose concentrations between control and β-cell-specific Gα(i2)-deficient mice. In contrast, the two- to threefold increase in insulin secretion evoked by L-arginine or L-ornithine (in the presence of 16 mM glucose) was significantly reduced in islets lacking Gα(i2). In accord with a reduced level of insulin secretion, intracellular calcium concentrations induced by the agonistic amino acid L-arginine did not reach control levels in β-cells. The presented analysis of gene-targeted mice provides novel insights in the role of β-cell Gα(i2) showing that amino acid-induced insulin-release depends on Gα(i2). Fil: Leiss, Veronika. University of Tübingen. Department of Pharmacology and Experimental Therapy; Alemania Fil: Flockerzie, Katarina. University of Tübingen. Department of Pharmacology and Experimental Therapy; Alemania Fil: Novakovic, Ana. University of Tübingen. Department of Pharmacology and Experimental Therapy; Alemania Fil: Rath, Michaela. German Institute of Human Nutrition. Department of Experimental Diabetology; Alemania Fil: Schönsiegel, Annika. University of Tübingen. Department of Pharmacology and Experimental Therapy; Alemania Fil: Birnbaumer, Lutz. National Institute ofEnvironmental Health Sciences. Laboratory of Neurobiology; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schürmann, Annette. German Institute of Human Nutrition. Department of Experimental Diabetology; Alemania Fil: Harteneck, Christian. University of Tübingen. Department of Pharmacology and Experimental Therapy; Alemania Fil: Nürnberg, Bernd. University of Tübingen. Department of Pharmacology and Experimental Therapy; Alemania

The presence of cGMP-dependent protein kinase I (cGKI) in murine adipocytes has been questioned, although cGKI was implicated in the thermogenic program of fat cells (FCs) and to exert anti-hypertrophic/-inflammatory effects in white adipose tissue. Herein, cGKI was detected in adipocytes from control mice, whereas FCs from global cGKI knockouts (cGKI−/−) and cGKIα rescue (αRM) mice remained cGKI-negative. cGKI mutants exhibit decreased adipocyte size, plasma leptin levels and reduced body-weights as compared to litter-matched controls. Low abundance of adiponectin in WAT and plasma of αRM animals together with previously confirmed high IL-6 levels indicate a low-grade inflammation. However, αRMs were protected from streptozotocin-induced hyperglycemia. Our results suggest that cGMP/cGKI affects both glucose and FC homeostasis in more complex mode than previously thought.

We have previously shown that activation of G i2, an subunit of the heterotrimeric G protein complex, induces skeletal mus-cle hypertrophy and myoblast differentiation. To determine whether G i2 is required for skeletal muscle growth or regenera-tion, G i2-null mice were analyzed. G i2 knockout mice display decreased lean body mass, reduced muscle size, and impairedskeletal muscle regeneration after cardiotoxin-induced injury. Short hairpin RNA (shRNA)-mediated knockdown of G i2 insatellite cells (SCs) leads to defective satellite cell proliferation, fusion, and differentiationex vivo. The impaired differentiationis consistent with the observation that the myogenic regulatory factors MyoD and Myf5 are downregulated upon knockdown ofG i2. Interestingly, the expression of microRNA 1 (miR-1), miR-27b, and miR-206, three microRNAs that have been shown toregulate SC proliferation and differentiation, is increased by a constitutively active mutant of G i2 [G i2(Q205L)] and counter-regulated by G i2 knockdown. As for the mechanism, this study demonstrates that G i2(Q205L) regulates satellite cell differen-tiation into myotubes in a protein kinase C (PKC)- and histone deacetylase (HDAC)-dependent manner. Fil: Minetti, Giulia C.. Novartis Campus. Basilea; Suiza Fil: Feige, Jerome N.. Campus EPFL. Lausania; Suiza Fil: Bombard, Florian. Novartis Campus. Basilea; Suiza Fil: Heier, Annabelle. Novartis Campus. Basilea; Suiza Fil: Morvan, Fredric. Novartis Campus. Basilea; Suiza Fil: Nürnberg , Bernd. University of Tübingen. Tübingen; Alemania Fil: Leiss, Veronika. University of Tübingen. Tübingen; Alemania Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Research Triangle Park. North Carolina; Estados Unidos Fil: Glass, David J. Novartis Institutes for Biomedical Research. Cambridge; Estados Unidos Fil: Fornaro, Mara. Novartis Campus. Basilea; Suiza

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Insulin and glucagon are the major hormones of the islets of Langerhans that are stored and released from the B- and A-cells, respectively. Both hormones are secreted when the intracellular cytosolic Ca2+ concentration ([Ca2+]i) increases. The [Ca2+]i is modulated by mutual inhibition and activation of different voltage-gated ion channels. The precise interplay of these ion channels in either glucagon or insulin release is unknown, owing in part to the difficulties in distinguishing A- from B-cells in electrophysiological experiments. We have established a single-cell RT-PCR method to identify A- and B-cells from the mouse. A combination of PCR, RT-PCR, electrophysiology and pharmacology enabled us to characterize the different sodium and calcium channels in mouse islet cells. In both A- and B-cells, 60% of the inward calcium current (I(Ca)) is carried by L-type calcium channels. In B-cells, the predominant calcium channel is Ca(v)1.2, whereas Ca(v)1.2 and Ca(v)1.3 were identified in A-cells. These results were confirmed by using mice carrying A- or B-cell-specific inactivation of the Ca(v)1.2 gene. In B-cells, the remaining I(Ca) flows in equal amounts through Ca(v)2.1, Ca(v)2.2 and Ca(v)2.3. In A-cells, 30 and 15% of I(Ca) is due to Ca(v)2.3 and Ca(v)2.1 activity, respectively, whereas Ca(v)2.2 current was not found in these cells. Low-voltage-activated T-type calcium channels could not be identified in A- and B-cells. Instead, two TTX-sensitive sodium currents were found: an early inactivating and a residual current. The residual current was only recovered in a subpopulation of B-cells. A putative genetic background for these currents is Na(v)1.7.