Developments in Molecular Pharmacology

The field of molecular pharmacology has advanced our understanding of excitable membrane proteins and their mechanisms of action. Before the advent of molecular biology, most pharmacology research used ligand binding and protein purification techniques to elucidate the structure and mechanism of action of various drugs and receptors. Although important, these studies were tedious because tissue contain more than one receptor subtype, and protein purification of receptors to homogeneity is a lengthy and complicated process. Molecular techniques provide a unique opportunity to study receptors and their structure at the DNA level. Many receptors and receptor subtypes have been discovered, paving the way for more development of receptor-specific pharmacologic agents. We briefly review the general field of molecular pharma-cology because future drugs for perioperative use probably will target receptor and receptor subtypes discovered using molecular approaches.

Molecular pharmacology takes advantage of the finding that all proteins, including excitable membrane proteins, are encoded in the human genome as nucleic acids. Every amino acid in a protein is encoded by a specific combination of three nucleotides in DNA. If the DNA sequence that encodes a receptor protein can be determined, the putative primary structure (i.e., amino acid sequence) of the receptor can be deduced. Encoding DNA sequences (i.e., genes) can be inserted into special cells that can express (i.e., manufacture, assemble, and deliver to the appropriate location in the cell) receptor protein in high quantity. This has several advantages.

First, studies on the receptor itself can be performed. By changing (i.e., mutating) nucleotide sequences, an abnormal (i.e., synthetic or "designer") receptor can be created. This abnormal receptor can then be compared with the original receptor to see whether the changes made affect binding of drug to the receptor or its coupling to second messengers. In this manner, the function of each portion of the receptor can be studied. This type of information is often called structure-activity relationships. G proteins and second messengers can be investigated in a similar manner. Naturally occurring human receptor variants have been investigated and tested for alterations in pharmacologic properties (see "Pharmacogenetics").

The second advantage of using molecular techniques in pharmacology is that new receptors and receptor subtypes can be discovered by searching the genome for DNA sequences similar to those of known receptors. After these receptors are discovered, they can be easily characterized, because high expression in cells enables them to be screened by various pharmacologic agents. The availability of DNA sequences for the full human genome has accelerated the discovery of receptors through DNA homology searching.

Perhaps the most immediately clinically relevant point is that new investigational drugs can be rapidly screened for effects on various native and variant receptors. In this way, pharmacologic effects of individual receptors can be studied in a controlled manner, isolated from other receptors and receptor subtypes. Such studies have the potential to lead to development of new drugs for use in the perioperative period and for understanding the effects of these drugs in patients with receptor variants.

Although there are many advantages to using molecular approaches to understanding physiologic pathways of individual receptors and in drug discovery, there are also some disadvantages. Cell lines are not integrated organisms, and the function of the receptor protein in a network of physiologic systems cannot be assessed. One approach to understanding the function of a previously identified

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The mouse model is robust and useful, but it is important to remember that this scientific model has its own limitations. To interpret data from transgenic and knockout animals correctly, at least three limitations of this technology should be considered. First, the genetic background of animals created must be carefully examined. If not carefully controlled, altered phenotypes may result from differences in animal strains (or genetic background) rather than from an overexpressed or knocked-out gene product. Second, elimination of specific receptors may be compensated for by alterations of other gene products. In this case, the final phenotype represents the net alteration of several genes; this is of particular concern when elimination of a gene is lethal because surviving progeny

TABLE 3-1 -- Results of targeting various portions of adrenergic-receptor signaling pathways in knock-out and transgenic mice, with an emphasis on cardiovascular effects

Model	Target	Result
Transgenic	↑β2 AR	↑Basal AC, ↑LV function, ↑atrial contractility, ↑supraventricular premature beats, ↓HR variability
Knock-out	↓β2 AR	No observable effects (except effects of mild exercise)
Transgenic	↑β1 AR	Dilated cardiomyopathy and early death; fibrosis
Knock-out	↓β1 AR	Prenatal death rate of 70%; among survivors: normal AC, ↓ISO-stimulated effects
Transgenic	↑AC-V	↑HR, ↑fractional shortening, no ISO-stimulated effects
Transgenic	↑GRK2	↓AC activity, ↓βARs function, ↓ISO-stimulated effects (rescued with βARK-ct peptide, which inhibits GRK2)
Transgenic	↑GRK5	Enhanced βAR desensitization but not angiotensin II desensitization
Transgenic	↑GRK2 inhibitor (↓GRK2 function)	Enhanced cardiac contractility with ISO
Knock-out	↓GRK2	Lethal phenotype, gestational LV hyperplasia, LVEF <70% in embryos
Transgenic	↑Gs α	No change in baseline EF, ↑ISO-stimulated effects, myocardial fibrosis
Knock-out	↓Phospholamban	↓βAR-mediated contractile responses
Transgenic	↑ α1a AR	Hypertension, ↑inotropy
Transgenic	↑ α1b AR (constitutively active)	Myocardial hypertrophy, hypertension, nociception, memory
Transgenic	↑ α1b AR (wild type)	No myocardial hypertrophy or hypertension
Transgenic	↑ α1d AR	Nociception, memory
Transgenic	↑Gq	Myocardial hypertrophy (about fourfold overexpression), higher expression produces heart failure
Transgenic	↑Gq inhibitor (↓Gq function)	Prevention of myocardial hypertrophy
Knock-out	↓α2a AR	Presynaptic α2 AR, mediates sedation and hypnosis, ↓BP (central hypotension), analgesia, regulation of DA/5-HT, antiepileptogenic effects of NE
Knock-out	↓α2b AR	Mediates ↑BP (peripheral vascoconstriction)
Knock-out	↓α2c AR	Mediates hypothermia, DA synthesis and metabolism, and presynaptic α2 AR (at low-frequency stimulation)
AC, adenylyl cyclase; AR, adrenergic receptor; βARK, β-adrenergic receptor kinase; BP, blood pressure; DA, dopamine; HR, heart rate; 5-HT, serotonin; ISO, isoproterenol; LV, left ventricle; G, G protein; GRK, G protein-coupled receptor kinase; LVEF, left ventricular ejection fraction; NE, norepinephrine; ↑, increased; ↓, decreased.

by definition have compensated in a way that enhances survival and therefore may not be representative of the results of a single-gene knock-out experiment. Some receptors and proteins have different tissue localizations in humans and other animal species. Results from transgenic and knock-out animal studies should be interpreted cautiously in this circumstance to prevent incorrect conclusions from being drawn regarding the implications for human physiology.

Despite these potential limitations, transgenic and knock-out animal models have proved very important in elucidating novel functions of receptors and proteins. Many unexpected physiologic roles for gene products have been discovered, and important physiologic questions have been answered and confirmed using these approaches. As an example, Table 3-1 lists conclusions drawn from transgenic and knock-out mice based on alterations of adrenergic receptors and their signal transduction cascade. Novel functions for specific adrenergic receptor subtypes (e.g., the role of α_2a -adrenergic receptors in sedation and CNS-mediated hypotension, α_2b -adrenergic receptors in mediating vasoconstriction, α_2c -adrenergic receptors in

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