As the central pacemaker, the suprachiasmatic nucleus (SCN) has long been considered the primary regulator of blood pressure circadian rhythm; however, this dogma has been challenged by the discovery that each of the clock genes present in the SCN is also expressed and functions in peripheral tissues. The involvement and contribution of these peripheral clock genes in the circadian rhythm of blood pressure remains uncertain. Here, we demonstrate that selective deletion of the circadian clock transcriptional activator aryl hydrocarbon receptor nuclear translocator–like (Bmal1) from smooth muscle, but not from cardiomyocytes, compromised blood pressure circadian rhythm and decreased blood pressure without affecting SCN-controlled locomotor activity in murine models. In mesenteric arteries, BMAL1 bound to the promoter of and activated the transcription of Rho-kinase 2 (Rock2), and Bmal1 deletion abolished the time-of-day variations in response to agonist-induced vasoconstriction, myosin phosphorylation, and ROCK2 activation. Together, these data indicate that peripheral inputs contribute to the daily control of vasoconstriction and blood pressure and suggest that clock gene expression outside of the SCN should be further evaluated to elucidate pathogenic mechanisms of diseases involving blood pressure circadian rhythm disruption.

Mice deficient in regulator of G-protein signaling-2 (RGS2) have severe hypertension, and RGS2 genetic variations occur in hypertensive humans. A potentially important negative feedback loop in blood pressure homeostasis is that angiotensin II (Ang II) increases vascular smooth muscle cell (VSMC) RGS2 expression. We reported that Group VIA phospholipase A₂ (iPLA₂β) is required for this response (Xie, Z., Gong, M. C., Su, W., Turk, J., and Guo, Z. (2007) J. Biol. Chem. 282, 25278–25289), but the specific molecular causes and consequences of iPLA₂β activation are not known. Here we demonstrate that both protein kinases C (PKC) and A (PKA) participate in Ang II-induced VSMC RGS2 mRNA up-regulation, and that actions of PKC and PKA precede and follow iPLA₂β activation, respectively. Moreover, we identified a conserved cAMP-response element (CRE) in the murine RGS2 promoter that is critical for cAMP-response element-binding protein (CREB) binding and RGS2 promoter activation. Forskolin-stimulated RGS2 mRNA up-regulation is inhibited by CREB sequestration or specific disruption of the CREB-RGS2 promoter interaction, and Ang II-induced CREB phosphorylation and nuclear localization are blocked by iPLA₂β pharmacologic inhibition or genetic ablation. Ang II-induced intracellular cyclic AMP accumulation precedes CREB phosphorylation and is diminished by inhibiting iPLA₂, cyclooxygenase, or lipoxygenase. Moreover, three single nucleotide polymorphisms identified in hypertensive patients are located in the human RGS2 promoter CREB binding site. Point mutations corresponding to these single nucleotide polymorphisms interfere with stimulation of human RGS2 promoter activity by forskolin. Our studies thus delineate a negative feedback loop to attenuate Ang II signaling in VSMC with potential importance in blood pressure homeostasis and the pathogenesis of human essential hypertension.

The cyclooxygenases (COX-1 and COX-2) generate prostaglandin H₂ from arachidonic acid (AA). In its catalytically productive conformation, AA binds within the cyclooxygenase channel with its carboxylate near Arg-120 and Tyr-355 and ω-end located within a hydrophobic groove above Ser-530. Although AA is the preferred substrate for both isoforms, COX-2 can oxygenate a broad spectrum of substrates. Mutational analyses have established that an interaction of the carboxylate of AA with Arg-120 is required for high affinity binding by COX-1 but not COX-2, suggesting that hydrophobic interactions between the ω-end of substrates and cyclooxygenase channel residues play a significant role in COX-2-mediated oxygenation. We used structure-function analyses to investigate the role that Arg-120 and residues lining the hydrophobic groove play in the binding and oxygenation of substrates by murine (mu) COX-2. Mutations to individual amino acids within the hydrophobic groove exhibited decreased rates of oxygenation toward AA with little effect on binding. R120A muCOX-2 oxygenated 18-carbon ω-6 and ω-3 substrates albeit at reduced rates, indicating that an interaction with Arg-120 is not required for catalysis. Structural determinations of Co³⁺-protoporphyrin IX-reconstituted muCOX-2 with α-linolenic acid and G533V muCOX-2 with AA indicate that proper bisallylic carbon alignment is the major determinant for efficient substrate oxygenation by COX-2. Overall, these findings implicate Arg-120 and hydrophobic groove residues as determinants that govern proper alignment of the bisallylic carbon below Tyr-385 for catalysis in COX-2 and confirm nuances between COX isoforms that explain substrate promiscuity.