Andrzej Chruscinski

Cardiovascular Regulation in Mice Lacking α2-Adrenergic Receptor Subtypes b and c

α2-Adrenergic receptors (α2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific α2AR subtypes (α2a, α2b, and α2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either α2b or α2c receptors. Stimulation of α2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating α2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the α2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective α2AR drugs.

Scalable signaling mediated by T cell antigen receptor–CD3 ITAMs ensures effective negative selection and prevents autoimmunity

The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

A genetically engineered cell-based biosensor for functional classification of agents

Cell-based biosensors (CBBs) utilize whole cells to detect biologically active agents. Although CBBs have shown success in detecting the presence of biological agents, efforts to classify the type of agent based on functional activity have proven difficult because multiple biochemical pathways can lead to the same cellular response. However, a new approach using a genetically-engineered cell-based biosensor (GECBB) described in this paper translates this cross-talk noise into common-mode noise that can be rejected. The GECBB operates by assaying for an agents ability to differentially activate two populations of cells, wild-type (WT) cells and cells genetically engineered to lack a specific receptor, knockout (KO) cells. Any biological agent that targets the knocked out receptor will evoke a response in the WT but not in the KO. Thus, the GECBB is exquisitely sensitive to agents that effect the engineered pathway. This approach provides the benefits of an assay for specific functional activity while simplifying signal analysis. The GECBB implemented was designed to be sensitive to agents that activate the beta 1-adrenergic receptor (beta 1-AR). This was achieved by using mouse cardiomyocytes in which the beta 1-AR had been knocked out. The cellular signal used in the GECBB was the spontaneous beat rate of the two cardiomyocyte syncitia as measured with microelectrode arrays. The GECBB was able to detect the beta-AR agonist isoproterenol (ISO) at a concentration of 10 microM (P<0.005).

Cholinergic Modulation of Angiogenesis: Role of the 7 Nicotinic Acetylcholine Receptor

Pathological angiogenesis contributes to tobacco‐related diseases such as malignancy, atherosclerosis and age‐related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except α2, α4, γ, and δ in four different types of ECs. Using siRNA methodology, we found that the α7 nAChR plays a dominant role in nicotine‐induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine‐activated EC functions (proliferation, survival, migration, and tube formation). The α9 and α7 nAChRs have opposing effects on nicotine‐induced cell proliferation and survival. Our studies reveal a critical role for the α7 nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis. J. Cell. Biochem. 108: 433–446, 2009.

Cloning and expression of the mouse homolog of the human α2-C2 adrenergic receptor

Summary Three subtypes of α2 adrenergic receptors have been identified in the human and rat. The subtype located on human chromosome 2 (α2-C2) is unique in that it is expressed mainly in the peripheral tissues and lacks sites for N-linked glycosylation. We isolated the gene encoding the mouse homolog of the human α2-C2 adrenergic receptor (Mα2-2H). The deduced amino acid sequence of the Mα2-2H shows 82% and 96% identity to the human α2-C2 and the rat RNGα2 adrenergic receptors, respectively. Southern blot analysis demonstrated that the Mα2-2H was encoded by a single copy gene and was distinct from the mouse homologs of the α2-C4 and α2-C10 adrenergic receptors. When expressed in COS-7 cells, the Mα2-2H exhibited a pharmacological profile similar to the human α2-C2 and rat RNGα2 receptors.

Primary structure of the mouse β1-adrenergic receptor gene

Abstract The mouse β 1 - adrenergic receptor was isolated from a genomic library and cloned into pBluescript SK − . Characterization of the clone revealed an open reading frame which encodes a predicted protein of 466 amino acids. The mouse β 1 receptor is 92.7% identical to the human sequence, 98.5% identical to the rat sequence, and contains a consensus site for N-linked glycosylation at Asn-15 and a cAMP-dependent protein kinase phosphorylation site at Ser-301.

Cardiovascular β-Adrenergic Receptor Subtype Physiology Studied by Targeted Gene Disruption • 95

Cardiovascular β-Adrenergic Receptor Subtype Physiology Studied by Targeted Gene Disruption • 95