Our group pursues three inter-related themes in basic and clinical approaches to autonomic and renal function:
Cell biology of catecholamine storage vesicles:
Implications for hypertension.
The sympathoadrenal efferent branch of the autonomic nervous system plays a key minute-to-minute role in regulation of blood pressure, and excessive sympathoadrenal activity is clearly implicated in the pathogenesis of hypertension, both primary (genetic, essential) and secondary (acquired), in both humans and experimental animals.
This system acts by co-release through exocytosis (all-or-none discharge) of co-transmitters from secretory vesicles of postganglionic sympathetic axons and chromaffin cells, into the bloodstream or neuroeffector junctions (synaptic clefts), wherein co-transmitters impinge on cardiovascular target cells, such as vascular smooth muscle, myocardiocytes, and endothelial cells, thereby regulating blood pressure.
While the best-studied sympathoadrenal co-transmitters are the catecholamines (norepinephrine and epinephrine), a complex "cocktail" of substances is co-released by exocytosis from storage vesicles, including not just the catecholamines themselves, but also neuropeptides such as several large acidic proteins, the chromogranins / secretogranins, which are cleaved to biologically active peptides which modulate both neurosecretion and vascular smooth muscle relaxation.
It has become clear that co-transmitters other than the catecholamines themselves also participate in the vascular responses to sympathoadrenal activation. Evidence in support of this principle includes the observation that the vasoconstriction of sympathetic activation is only partially reversed by alpha- (even in combination with beta-) adrenergic blockade. Both pre- and postsynaptic function may be disturbed in hypertension.
Increasingly, we have turned to the tools of genome technology and statistical genetics in order to understand how heredity shapes human functional responses in the sympathetic neuroeffector junction. We have already discovered substantial polymorphism at "candidate" genetic loci whose products maintain the activity of the neuroeffector junction (CHGA, CHGB, PNMT), and have found functional differences among allelic variants that predict autonomic (including stress blood pressure) responses in humans. A central theme of this program is discovery (by human genomic DNA resequencing) of the spectrum of human allelic variation at "candidate" genetic loci encoding proteins governing sympathetic neuroeffector function, and then subjecting these variants to functional testing, both by allelic and haplotype associations in vivo, and by expression studies in vitro. These complementary approaches allow us to determine how such variants might influence autonomic activity, both in human responses and at the level of their actions in isolated cells. Incorporation of newly emerging proteomic tools further allows us to understand how protein products of the target (candidate) genes finally undergo post-translational modification to accomplish their tasks in the junction.
Sympathoadrenal catecholamine secretion is exocytotic (all-or-none), releasing not just catecholamines but also the acidic proteins with which catecholamines are stored: chromogranins/secretogranins, quantitatively major components being chromogranin A (CHGA) and chromogranin B (CHGB). Both CHGA and CHGB seem to be necessary factors ("on/off switches") in the biogenesis of catecholamine secretory vesicles. CHGA is cleaved to biologically active fragments, including the endogenously formed "catestatin" that inhibits catecholamine release; its specific inhibitory mechanism seems to be nicotinic cholinergic antagonism.
Data collected in the past few years include discovery of substantial and functional human genetic diversity (polymorphism) at the CHGA and CHGB loci, including 3 novel variants of catestatin with differential potency as nicotinic antagonists, and 8 common CHGA proximal promoter SNPs, giving rise to common CHGA promoter haplotypes with different transcriptional activities in chromaffin cells. Common CHGA promoter polymorphisms predict plasma CHGA concentration. Common 3'-UTR polymorphisms in both CHGA and CHGB predict heritable variation in stress blood pressure responses in human twins. CHGB expression cosegregated with a novel locus on chromosome 11q24-q25, suggesting the presence of a previously uncharacterized major gene regulating human sympathetic outflow.
We will therefore emphasize effects of common human genetic variation (polymorphism) at the CHGA and CHGB loci on their biosynthesis, release, cellular targets, and autonomic consequences. We will characterize the inhibitory effects of catestatin variants on nicotinic cholinergic receptors and their signal transduction. We will explore whether polymorphisms at CHGA/catestatin and CHGB influence catecholamine release and autonomic physiology in vivo or in vitro. To explore the significance of such polymorphisms, we formulate questions that give rise to testable hypotheses that can be confirmed or refuted by crucial experiments. The studies are designed to shed light upon heritable regulation of nicotinic receptor-mediated release of catecholamines, and their consequences for autonomic control of the circulation.
Pharmacogenetic influences on autonomic drug responses in the cardiovascular system: Autonomic Pharmacodynamic Pharmacogenomics.
We have developed a center devoted to uncovering how allelic variation in genetic loci encoding autonomic junctional receptors, and their post-receptor signal-transducing molecules, determines cardiovascular drug responses in humans. We will focus on drug responses that influence pre- and post-synaptic receptor-mediated autonomic events in the cardiovascular system, including regulation of catecholamine storage and release, heart rate, and regional vascular tone. The disease targets, in which these autonomic drugs are used therapeutically, include systemic and pulmonary hypertension, heart failure, arrhythmias, renal failure, and edema (sodium and water retention).
Hypothesis and strategy. Determinants of drug action can be broadly divided into two areas: pharmacokinetics (disposition of the drug and access to the target site) and pharmacodynamics (mode and intensity of drug action at the target site). Our pharmacogenomics center probes pharmacodynamic determinants of human drug responses. Our focus is human autonomic cardiovascular drug responses, in both the systemic and pulmonary circulations. To direct this focus to pharmacodynamics of autonomic cardiovascular responses, our initial phenotyping strategy will emphasize regional (rather than systemic) vascular responses, so as to remove pharmacokinetic and baroreflex variables from their confounding influence, and thereby to isolate and focus upon pharmacodynamic (receptor, post-receptor, and effector) determinants of drug responses. The regional responses are characterized in the forearm and hand circulatory beds. Both pre-synaptic and post-synaptic responses are studied. This center evaluates both diagnostic and therapeutic drug responses. Diagnostic drug responses will allow us to more effectively and "cleanly" probe receptor and post-receptor signaling, thereby providing rigorous pharmacodynamic parameters to be applied later to therapeutic drug responses in humans. Variation in responses to exogenous agonist should be specific to variation (either qualitative or quantitative) in receptor, post-receptor signaling, or effector components; by contrast, variation in antagonist responses could represent changes in the amount of the endogenous agonist present. Diagnostic responses should allow definition of which SNP alleles contribute to variation in signaling in a particular pathway. We hypothesize that agonist responses, obtained under carefully defined circumstances in regional circulatory beds (pulmonary, renal, forearm, hand), will lead to the discovery of associated SNP alleles which will ultimately yield diagnostic tools to predict therapeutic drug responses (in particular, for antagonists at the same receptors). The functional significance of discovered SNP alleles is verified in vitro and in experimental organisms. Finally, such SNP alleles will then be tested in human trials of therapeutic drug responses to answer the question: Does SNP stratification predict therapeutic drug responses in large, prospective, randomized, controlled clinical trials?
In our laboratory, we systematically probe the human autonomic pharmacology of pre-synaptic catecholamine storage and release, as well as its post-synaptic consequences, using an isolated vascular bed (the human forearm, with local drug infusion, measurement of local neurotransmitter release, and local plethysmographic blood flow). This phenotyping strategy therefore emphasizes regional vascular responses, so as to remove pharmacokinetic and baroreflex variables from their confounding influence, to thereby isolate and focus upon pharmacodynamic (receptor and post-receptor) determinants of drug responses. The diagnostic pre-synaptic drug responses we propose (b2-adrenergic agonist, a2-adrenergic agonist, and sympathomimetic amine) will allow us to more effectively and "cleanly" probe receptor and post-receptor signaling, allowing rigorous pharmacodynamic insights to be applied later to therapeutic drug responses in humans. Diagnostic responses should allow definition of which SNP alleles contribute to variation in signaling in a particular pathway. Here we hypothesize that pre-synaptic agonist responses, obtained under carefully defined circumstances in a regional circulatory bed (forearm), will lead to the discovery of associated SNP alleles which will ultimately yield diagnostic tools to predict therapeutic drug responses (to antagonists at the same receptors).
Human twin studies of autonomic and renal function.
The overall goal of this project is to use the power of twin and sibling pairs, coupled with a dense microsatellite genome scan, to position loci, allelic variation at which influences autonomic function in either the sympathetic or parasympathetic branches.
The autonomic nervous system is the key second-to-second regulator of the circulation, and hence of blood pressure. Autonomic activity is deranged not only in patients with established hypertension, but also in their still-normotensive offspring; thus, autonomic traits are valuable as "intermediate phenotypes" in hypertension, a common disease with non-Mendelian (complex) inheritance.
Our specific aims are threefold:
2. Allelic association. At candidate loci, establish whether particular SNP alleles (or SNP haplotypes) associate with the intermediate phenotype, and later with the ultimate disease trait.
3. Genome-wide approaches (linkage). In sibling pairs derived from nuclear families ascertained on the basis of twins, couple intermediate phenotypes with genome-wide linkage approaches to position novel, previously unsuspected genetic loci that contribute to trait variation in the intermediate phenotypes.