Lutz Hein

Central Hypotensive Effects of the α2a-Adrenergic Receptor Subtype

α2-Adrenergic receptors (α2ARs) present in the brainstem decrease blood pressure and are targets for clinically effective antihypertensive drugs. The existence of three α2AR subtypes, the lack of subtype-specific ligands, and the cross-reactivity of α2AR agonists with imidazoline receptors has precluded an understanding of the role of individual α2AR subtypes in the hypotensive response. Gene targeting was used to introduce a point mutation into the α2aAR subtype in the mouse genome. The hypotensive response to α2AR agonists was lost in the mutant mice, demonstrating that the α2aAR subtype plays a principal role in this response.

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.

Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission.

The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. α2-Adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous α2-adrenergic-receptor subtypes (α2A, α2B, α2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three α2-adrenergic-receptor subtypes were disrupted. Here we show that both the α2A- and α2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. α2A-Adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the α2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both α2A- and α2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.

Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome

We report the existence of two structurally distinct forms of the angiotensin receptor AT-1 in the mouse. A Balb/c mouse genomic library was screened by homology screening with a polymerase chain reaction (PCR) amplified probe. Restriction mapping and sequencing of the isolated genes revealed the presence of two receptor isoforms, here named the mouse AT-1a and AT-1b receptors, containing 22 different amino acids. Receptor binding studies performed on COS-7 cells transfected with the two receptors revealed that they had similar binding profiles for angiotensin II, angiotensin III and AT-1 or AT-2 specific antagonists. Because many of the structural differences were in the carboxy terminal putative intracellular domain, we speculate that these isoforms may differ in their regulation, signal transduction, or desensitization mechanisms.

Common Genomic Response in Different Mouse Models of β-Adrenergic–Induced Cardiomyopathy

Background—Although &bgr;-adrenergic receptor (AR) blockade therapy is beneficial in the treatment of heart failure, little is known regarding the transcriptional mechanisms underlying this salutary action. Methods and Results—In the present study, we screened mice overexpressing Gs&agr;, &bgr;1AR, &bgr;2AR, or protein kinase A to test if a common genomic pathway exists in different models with enhanced &bgr;-adrenergic signaling. In mice overexpressing Gs&agr;, differentially expressed genes were identified by mRNA profiling. In addition to well-known markers of cardiac hypertrophy (atrial natriuretic factor, CARP, and &bgr;-myosin heavy chain), uncoupling protein 2 (UCP2), a protein involved in the control of mitochondrial membrane potential, and four-and-a-half LIM domain protein-1 (FHL1), a member of the LIM protein family, were predicted to be upregulated. Upregulation of these genes was confirmed by quantitative reverse transcriptase–polymerase chain reaction at all time points tested during the development of cardiomyopathy in mice overexpressing Gs&agr;. In mice overexpressing &bgr;1AR, &bgr;2AR, or protein kinase A, increased UCP2 and FHL1 expression was also observed at the onset of cardiomyopathy. &bgr;AR blockade treatment reversed the cardiomyopathy and suppressed the increased expression of UCP2 and FHL1 in mice overexpressing Gs&agr;. Conclusions—UCP2 and FHL1 are important candidate genes that correlate with the development of &bgr;AR-induced cardiomyopathy in different mouse models with enhanced &bgr;AR signaling. In addition to preserving cardiac function, &bgr;AR blockade treatment also prevents the genomic regulation that correlates with the onset of heart failure.

Pulmonary Hypertension and Right Heart Failure in Pituitary Adenylate Cyclase–Activating Polypeptide Type I Receptor–Deficient Mice

Background—Pituitary adenylate cyclase–activating polypeptide (PACAP), acting via 3 different G protein–coupled receptors, has been implicated in the regulation of several homeostatic systems in the body, including cardiopulmonary control. To define the physiologic role of the PACAP-preferring type I receptor, PAC1, in cardiopulmonary function, we developed a mutant mouse strain lacking functional PAC1 receptors. Methods and Results—When PAC1-deficient mice were crossed onto a C57BL/6 background, almost all mutants died during the second postnatal week. Whereas mutant mice were indistinguishable from their wild-type littermates at birth, they showed progressive weakness and died from rapidly developing heart failure. Right ventricles of PAC1 mutants were massively dilated and showed cardiac myocyte hypertrophy, whereas left ventricular structure was unaltered. On direct cardiac catheterization, right ventricular pressure was elevated by 45% in PAC1-deficient mice, indicating increased pulmonary artery pressure, as no malformations were detected in the valves or outflow tract of the right ventricle. Consistent with elevated pulmonary pressure, lung capillary density was decreased by 30% and small pulmonary arteries of mutant mice had significant vascular smooth muscle cell hypertrophy compared with wild-type mice. Conclusions—Whereas PACAP induces vasodilation in isolated pulmonary vessels in wild-type mice, the absence of its specific receptor PAC1 causes pulmonary hypertension and right heart failure after birth. These in vivo findings demonstrate the crucial importance of PAC1-mediated signaling for the maintenance of normal pulmonary vascular tone during early postnatal life.

Modulation of vascular development and injury by angiotensin II

OBJECTIVE To examine the exact profile of expression and to determine the functional significance of the angiotensin II (Ang II), type I (AT1) and type 2 (AT2) receptors during rat aortic development and following rat carotid artery balloon injury. METHODS AT1 and AT2 mRNA levels in rat aortae were measured using a quantitative reverse transcription polymerase chain reaction technique. Ang II receptor function was assessed by quantitating the effects of AT1 (DuP753) and AT2 (PD123319) receptor antagonists during these processes. RESULTS During aortic development, AT1 expression was detected on gestational day 14, increased until embryonic day 16 (E16), after which, levels were similar throughout postnatal development. Conversely, AT2 mRNA first appeared at E16, reached maximal levels between E19 and neonatal day 1, and decreased thereafter. DNA synthesis rates decreased with aortic development (high at E15, 73.8 +/- 3.1%; dropping to 37.5 +/- 2.3% by E21). Whereas AT1 receptor antagonism accelerated this developmentally regulated decrease in DNA synthesis. AT2 receptor antagonism blunted this decrease. Because activated adult medial smooth muscle cells express a neonatal phenotype after vascular injury, we assessed Ang II receptor levels and function after carotid artery balloon injury. Both receptor subtypes increased; however, AT2 receptor mRNA expression peaked earlier than AT1 (48 to 72 h after injury). As with aortic development, DNA synthesis occurring between 24 to 48 h after injury (when AT2 receptors constitute 10% of the Ang II receptor population) decreased in DuP753-treated animals and increased in PD123319-treated animals. CONCLUSION These results indicate that Ang II receptors play a role in vascular development by promoting opposing effects on vascular smooth muscle cell growth.

Adrenergic Receptors From Molecular Structure to in vivo function

Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. Although several hundred G-protein-coupled receptors have been identified, adrenergic receptors, along with the visual pigment rhodopsin, have been among the most extensively studied members of this family of receptors. This review focuses on recent advances in understanding the molecular structure, function, and regulation of adrenergic receptors using in vitro systems and integrates recent transgenic animal models that were generated to study the adrenergic system in vivo. (Trends Cardiovasc Med 1997;7:137-145).

Vascular smooth muscle cell phenotype influences glycosaminoglycan composition and growth effects of extracellular matrix

Rat neonatal and neointimal vascular smooth muscle cells differ dramatically from adult medical vascular smooth muscle cells in their growth properties, with the neonatal and neointimal cells exhibiting growth in the absence of exogenously added growth factors. Since it has been hypothesized that extra-cellular matrix proteoglycans may influence the growth and differentiation of vascular smooth muscle cells, we examined the ability of matrix derived from these cells to influence vascular smooth muscle cell proliferation. To produce test matrices, cells were grown to confluence and removed by brief alkali treatment. Test cells were seeded onto these matrices and the rates of growth in a growth-factor-deficient medium determined. Compared to plastic wells, matrix from neonatal or neointimal cells stimulated the growth of vascular smooth muscle cells. Interestingly, matrix from adult cells was less efficient at promoting growth. Enzymatic digestion of extracellular matrix heparan sulfate, but not of other glycosaminoglycans, further increased the growth-stimulatory effect of extracellular matrix, suggesting that matrix heparan sulfate acts as a growth inhibitor. Consistent with this, biochemical analysis showed that the adult matrix contained a higher percentage of heparan sulfate compared with neonatal or neointimal matrix. These results suggest that autocrine production of heparan sulfate proteoglycans may play an important role in growth regulation of vascular smooth muscle cells during normal vascular development and differentiation as well as in pathological response to injury.