The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M1 receptor and represents a promising therapeutic strategy for cognitive disorders.

Transforming growth factor ␤ (TGF-␤) is implicated in the regulation of smooth muscle cell (SMC) differentiation. We previously identified a novel TGF-␤ control element (TCE) in the promoters of SMC differentiation marker genes, including ␣-smooth muscle actin and SM22␣. In this study, the importance of the TCE in regulation of SM22␣ gene expression in vivo was investigated by mutating it within the context of a mouse SM22␣ promoter-lacZ transgenic construct. Mutation of the TCE completely abolished SM22␣ promoter activity in arterial SMCs as well as in developing heart and skeletal muscle. To identify the transcription factor(s) binding to the TCE, we performed yeast one-hybrid cloning analysis and identified gut-enriched Krü ppellike factor (GKLF). However, cotransfection studies in cultured cells showed that GKLF repressed the TGF-␤-dependent increases in SM22␣ and ␣-smooth muscle actin promoter activities. Furthermore, GKLF was not highly expressed in differentiated SMCs in vivo, and TGF-␤ down-regulated GKLF expression in dedifferentiated cultured SMCs. In contrast, overexpression of a related factor (BTEB2) transactivated SM22␣ promoter activity. Thus, our findings suggest a reciprocal role for related Krü ppel-like transcription factors in the regulation of SMC differentiation through a TCE-dependent mechanism.

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Erk5 is a mitogen-activated protein kinase, the biological role of which is largely undefined. Therefore, we deleted the erk5 gene in mice to assess its function in vivo. Inactivation of the erk5 gene resulted in defective blood-vessel and cardiac development leading to embryonic lethality around embryonic days 9.5-10.5. Cardiac development was retarded largely, and the heart failed to undergo normal looping. Endothelial cells that line the developing myocardium of erk5؊͞؊ embryos displayed a disorganized, rounded morphology. Vasculogenesis occurred, but extraembryonic and embryonic blood vessels were disorganized and failed to mature. Furthermore, the investment of embryonic blood vessels with smooth muscle cells was attenuated. Together, these data define an essential role for Erk5 in cardiovascular development. Moreover, the inability of Erk5-deficient mice to form a complex vasculature suggests that Erk5 may play an important role in controlling angiogenesis.

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [ 3 H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.Smooth muscle cell (SMC) proliferation and migration are essential features of vasculogenesis and blood vessel maturation and clearly play a role in the pathophysiology of several prominent cardiovascular disease states, such as atherosclerosis, restenosis following balloon angioplasty, and hypertension (15,24). These processes are regulated by a number of humoral factors, including growth factors (i.e., platelet-derived growth factor [PDGF] and fibroblast growth factor), contractile agonists (i.e., angiotensin II and thrombin), and cytokines (i.e., transforming growth factor ␤ and interleukins) (1,15,24). Extracellular matrix (ECM) interactions are also important, and it is clear that deposition of ECM components within the blood vessel wall and the specific expression of SMC integrin receptors are regulated during vascular development and under pathologic conditions (27). Thus, it is important to identify the molecular mechanisms by which growth factor-and ECMmediated signaling pathways in SMCs converge to regulate proliferation and migration of vascular SMCs.Activation of the integrin families of transmembrane cell surface receptors is mediated by contact with ECM components such as fibronectin and collagen and leads to the formation of focal adhesion structures. Focal adhesions contain not only cytoskeletal components that physically tether the integrin cytoplasmic tail to the actin cytoskeleton but also a number of signaling molecules that are activated in ...