PHA 622

RECEPTOR REGULATION

AND CARDIOVASCULAR FUNCTION

 

 

            Recommended Reading and References

                                                                                               

Barki-Harrington, L., Perrino, C. And Rockman, H. ; Network integration of the adrenergic system in cardiac hypertrophy. Cardiovas. Res., 63; 391-402, 2004.

 

Bers, D.; Cardiac excitation-contraction coupling. Nature, 415; 198-205, 2002.

 

Dodge-Kafka, K., Langeberg, L., and Scott, J. ; Compartmentation of cyclic nucleotide signaling in the heart: The role of A-kinase anchoring proteins.  Cir. Res., 98; 993-1001, 2006.

 

Furchgott, R.F., The Classification of Adrenoceptors. An Evaluation from the Standpoint of Receptor Theory,, In, Catecholamines, ed, H. Blaschko and E. Muscholl, Springer Verlag

                                                           

Korzick, D ; Regulation of cardiac excitation-contracton coupling: A cellular update.

Adv. Physiol. Edu., 27; 192-200, 2003.

 

Lohse, M., Engelhardt, S. And Eschenhagen, T. ; What is the role of beta-adrenergic signaling in heart failure? Cir. Res., 93; 896-906, 2003

 

Pavoine, C. And Defer, N.; The cardiac beta2-adrenergic signalling a new role for cPLA2. Cel. Sig., 17; 141-152, 2005.

 

Perrino, C., Rockman, H. And Chiariello, M. ; Targeted inhibition of phosphoinositdie 3-kinase activity as a novel strategy to normalize beta-adrenergic receptor function in heart failure. Vas. Pharmacol., 45, 77-85, 2006. 

 

Ruffolo, R.R., Jr. Important Concepts of Receptor Theory, J. Autonomic Pharmacology 2 277-295

 

Steinberg, S.; Beta2-Adrenergic receptor signaling complexes in cardiomyocyte caveolae/lipid rafts.  J. Mol. Cell. Cardio.,  37; 407-415, 2004.

 

Zheng, M., Zhu, W., Han, Q and Xiao, R. ; Emerging concepts and therapeutic implications of beta-adrenergic receptor subtype signaling.  Pharmacol. And Ther. 108;  257-268, 2005.

 

 

 

The student should be able to explain or describe;

 

 

1.         The pharmacodynamic principles that aid in the understanding of adrenergic receptors and the actions of drugs on these receptors.

 

2.         The criteria upon which alpha and beta receptors are defined.

 

 

3.         The second messenger systems utilized by alpha and beta receptors and how activation of these receptors leads to a change in physiologic function.

 

 

4.         The effects of alpha and beta receptor activation on the heart and blood vessels.

 

5.         The effects of isoproterenol, epinephrine and norepinephrine on the cardiovascular system.

 

 

 

 

Key drugs

Isoproterenol - Isuprel

Epinephrine - Adrenalin

Norepinephrine- Levophed

 

 

 

 

 

 

 

The adrenergic receptors which subserve the responses of the sympathetic nervous system have been divided into two discrete subtypes: alpha adrenergic receptors (alpha receptors) and beta adrenergic receptors (beta receptors).  The classification of these receptors, and indeed receptors in general,  is based on the interaction of agonists and antagonists with the receptors.

 

 

 

Beta Receptors

 

 

Beta-adrenergic receptors are currently categorized into three subtypes: beta1, beta2, and beta3.

The physiologic role of the beta3 receptor is not as well defined as are those of the beta1- and beta2- adrenergic receptors. 

Selective agonists and antagonists of the beta3-adrenergic receptor have been developed. Activation of the beta3-adrenergic receptor results in a negative inotropic effect.  Activation of the cardiac beta3-adrenergic receptor results in an increase in heart rate. Similar to the b2-adrenergic receptor activation of the beta3-adrenergic receptor results in vasodilation.

The beta 3-adrenergic receptor is also being investigated for the treatment of an over active baldder.  

Activation of the beta3-adrenergic receptor has been shown to stimulate lipolysis, and a number of beta3-receptor–selective agonists have been developed and are effective in rodent models of obesity.

Thus far none of the selective beta3-receptor agonists has been shown to be effective in stimulating weight loss in human beings.

 

 

 

 For the purposes of this material we will focus on the beta1 and beta2 receptors only.  The classification of beta receptors is based on the interaction of a series of drugs with these receptors.  The ability of epinephrine, norepinephrine and isoproterenol to increase the force of myocardial contraction was examined and the dose-response curves shown below were obtained.  Equilibrium dissociation constants for these ligands were  ISO, 80 nm,  E, 800 nM, and NE, 1000 nM.  Thus, the rank order of affinities for the beta receptor in the heart  is ISO>E>NE.   A beta receptor with these characteristics is  referred to as a beta1 receptor.

 

 

 

                                                                                                

 

 

 

The ability of the same compounds to produce bronchodilation was examined and a different set of dose response curves and equilibrium dissociation constants were obtained. The dissociation constants were  ISO, 80 nm,  E, 800 nM, and NE, 10,000 nM.   Notice how the ability to activate the beta receptors is dependent on the structure of the drugs under study.  Clearly then the receptor in the lung is different from that in the heart and is referred to as a beta2 receptor. These examples serve to illustrate how the equilibrium dissociation constant is often used as a “finger print” to identify a receptor.  Regardless of its location, the same receptor will have the same dissociation constants for agonists and antagonists.  As shown here the differences in equilibrium dissociation constants is an indication of heterogeneity within the main receptor population.   As will be illustrated throughout the course, receptor subtypes are routinely exploited in drug development to make ligands that interact selectively with one subtype in preference to another.

 

 

 

Beta Receptor Systems

 

 

Most tissues express multiple receptors.  However, the receptor mainly utilized by the sympathetic nervous system to affect myocardial function in the normal heart is the beta1 receptor; while in vascular and nonvascular smooth muscle it is the beta2 receptor. 

 

 

Tissue Receptor Subtype
Heart beta1
Adipose tissue beta1beta3?
Vascular Smooth Muscle beta2
Airway Smooth Muscle beta2
Kidney-renin release from JG cells beta1

 

 

THE BETA1 SYSTEM

 

 

Mechanism of Beta1 Receptor Activation in Cardiac Muscle

           1)  Agonist binds to the myocardial beta1-adrenergic receptor.  This receptor is a typical G-protein coupled receptor.

           

  2)  In the unstimulated state the G-protein is complexed with GDP.

 

  3) The receptor promotes exchange of GTP for GDP and release of G"/GTP.

 

  4) The G"/GTP complex activates adenylate cyclase.

 

  5) Intracellular cAMP increases and activates cAMP dependent protein kinase (PKA).

 

  6)  PKA phosphorylates the Ca2+ channel promoting Ca2+ influx.

 

  7)  Intracellular Ca2+ increases activating the contractile proteins.

 

  8) PKA phosphorylates the sarcoplasmic reticulum leading to an increase in Ca2+  uptake and release.

 

  9) PKA phosphorylates troponin changing its calcium binding kinetics.

 

  10)   Prolonged stimulation can lead to receptor down regulation via PKA and other protein kinases which phosphorylate the receptor.  The other protein kinases which are involved in phosphorylation are referred to as G-protein coupled receptor kinases or GRKS.  These phosphorylation steps lead to internalization of the receptor.

 

 

 

 

 

 

 

Cellular Signaling Activated by the  Beta Receptor in the Heart

 

 

Activation of the beta1 receptor leads to  increases in contractile force and heart rate.  The increase is myocardial contraction is a result of activation of those beta receptors associated with the atria and ventricle (especially the ventricles) while the increases in rate of contraction are due to activation of those receptors associated with the SA and AV nodes as well as the His-Purkinjie system.  Recall that the primary ion channels in the SA and AV nodes are calcium channels while in the His-Purkinjii and ventricular myocardium the electrical current is carried by sodium channels.  Activation of the beta receptor increases ion movements through both types of channels.  These actions result in an increase in heart rate.

 

            1)  Increase slope of phase 4 spontaneous depolarization

 

            2)  Increase maximal rate of phase 0 depolarization

 

            3)  Increase conduction velocity

 

            4)  Decrease refractory period

 

These electrophysiologic factors contribute to the orderly, rhythmic electrical activity that assures the efficient contractile activity of the heart.  In response to beta receptor activation, these parameters increase and the heart beats at a faster rate.  However, excess stimulation of the beta receptor by catecholamines can enhance these variables to such an extent that arrhythmias can occur.   Rhythm disturbances are a major concern with drugs that activate the beta1 receptor.   Drugs to be covered that have a tendency to generate arrhythmias include epinephrine, isoproterenol, norepinephrine, dopamine and dobutamine.

 

 

 

TheBeta1-Adrenergic Receptor as a Therapeutic Target

 

Agonists- congestive heart failure

Antagonists- hypertension, ischemic heart disease, congestive heart failure


 

 

Regulation of Receptor Function

 

 

Continuous exposure of an agonist results in a phenomenon referred to as desensitization.  The same concentration of agonist becomes less and less effective at producing the same level of effect.  Recent evidence has suggested potential mechanisms by which desensitization occurs.  The receptor becomes phosphorylated in the third cytoplasmic loop and c-terminal tail.  The phosphorylated receptor is less efficient at activating G-protein and also exhibits lower affinity for agonists.  The receptors can also be removed from and sequestered away from the cell surface.  These events indicate that second messengers not only regulate intracellular processes but are also capable of regulating the receptor systems which generate them.

 

 

 

 

 

 

 

 

 

Effect of Beta2 Receptor Activation on Smooth Muscle

 

 

The beta2 receptor associated with smooth muscle also utilizes the cAMP signaling system.  However, the results of receptor mediated increases in cAMP levels in smooth muscle are different than those occurring in cardiac muscle.  Therefore, the consequences of PKA phosphorylation of key structures in smooth muscle lead to relaxation                  

 

Structures Phosphorylated in Smooth Muscle

                                                                                   

 

            1)  sarcolemma  - Decrease Ca2+ influx

 

            2)  sarcoplasmic reticulum - Enhance Ca2+ uptake

 

            3)  decrease actin-myosin interactions - muscle relaxation

 

The net result of these activities is to inhibit calcium pathways in smooth muscle leading to relaxation.

 

 

 

 

 

 

 

 

 

 

ALPHA RECEPTORS SYSTEMS

 

If the ability of isoproterenol, epinephrine and norepinephrine to produce constriction of vascular smooth muscle is studied, the following dose-response curves and equilibrium dissociation constants were obtained: E, 5 uM, NE, 6, uM and ISO, 1000 uM.  You should begin to understand the reasons why the receptor causing vasoconstriction MUST be different from that causing cardiac contraction or bronchodilation. 

 

 

 

 

The receptor that produces vasoconstriction is referred to as an alpha receptor (affinity rankings of  E $ NE >>>ISO).  Observe how the structure of each drug affects that ability of these ligands to activate the alpha receptor. The concentration of isoproterenol necessary to activate alpha receptors is so large that isoproterenol can be thought of as a pure beta receptor agonist.

 

Alpha receptors have also been subdivided into alpha1 and alpha2 receptors. Epinephrine and norepinephrine have equal affinity at both alpha1 and alpha2 receptors.  However, other drugs were found to have higher affinity for one receptor over another and these differences in affinity were the evidence used to subclassify the receptors into alpha1 and alpha2.   More recently, three subtypes of the alpha1-receptor, the alpha1A,  alpha1B and alpha1D have been isolated, cloned and characterized.  Similarly, 3 subtypes of the alpha2-receptor, the alpha2A,  the alpha2B and the alpha2C have also been identified.  There is little doubt that these receptor subtypes subserve different physiologic functions. 

 

 

 

 

 

Mechanism of Alpha1 Receptor Activation of Smooth Muscle Contraction

 

 

In the case of the postjunctional alpha1 receptor, the inositol phosphate/diacylglycerol signaling pathway is activated by receptor occupancy. 

 

1)   Agonist binds to the vascular smooth muscle alpha1-receptor.  The receptor is a  typical G-protein coupled receptor with 7 membrane spanning regions.

 

          2)   In the unstimulated state the G-protein is complexed with GDP.

 

          3)   The receptor promotes exchange of GTP for GDP and release of G"/GTP.

 

          4) The G-protein activates phospholipase C leading to an increase in the intracellular second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG).

          5)  IP3 binds to specific sites on the SR and stimulates the release of intracellular Ca2+.

 

          6) Ca2+ influx is activated.

 

          7)  Like the beta-receptors, alpha receptors can also be desensitized and down regulated via phosphorylation of the receptor.

 

 

Effect of Epinephrine on Vascular Smooth Muscle

 

Associated with vascular smooth muscle are a large number of alpha1 receptors relative to beta2 receptors.  However, epinephrine has a higher affinity for the beta2 receptor relative to the alpha1 receptor (see above).  Activation of the beta2 receptor would produce vasodilation while activation of the alpha1 receptor would result in vasoconstriction.    Therefore, the effect of epinephrine on smooth muscle is dependent on its relative affinity for alpha1 and beta2  receptors and its concentration.    At low doses, epinephrine can selectively stimulate beta2 receptors producing muscle relaxation and a decrease in peripheral resistance.  However, once epinephrine concentrations are reached that bind to the alpha1 receptor, vasoconstriction will occur.  The two effects (smooth muscle relaxation and contraction) will oppose one another.

 

 

 

Effects of Norepinephrine and Isoproterenol on Smooth Muscle

 

 

Recall that norepinephrine in physiologically relevant concentrations has little affinity for beta2 receptors.  Therefore, it will stimulate only alpha1 receptors producing an increase in peripheral vascular resistance.  In contrast, the lack of activity at the alpha1-receptor means that isoproterenol will produce only a beta2-receptor mediated vasodilation.

 

Other Cardiovascular Functions

 

Alpha1 receptors also exist on the myocardium.  These receptors increase force without affecting rate.  The role of these receptors in physiologic regulation of myocardial performance or as a site of drug action has not be clearly elucidated.

 

Effects on the Cardiovascular System

 

For the drugs listed below, indicate how the drugs would affect (increase, decrease, no changes) heart rate, contractile force, total peripheral resistance (TPR) and systemic arterial blood pressure.  Recall the equations below.  Remember also that the effectors in the cardiovascular system (brain, kidney, heart and blood vessels) are all involved in the integrated  regulation of blood pressure. 

 

Blood pressure  =  Cardiac Output x TPR

Cardiac output  =  Stroke Volume x Heart Rate

Blood pressure = (Stroke volume x Heart rate) x Total peripheral vascular resistance

 

 

Completed Table Heart
Rate
Contractile
Force
TPR Blood
Pressure
Isoproterenol        
Norepinephrine        
Low Doses of Epi

**sufficient to activate the beta2, but not the alpha1 receptor.

       
High Doses of Epi

**sufficient to activate the alpha1

       

 

Applications to Therapeutics

 

Oral dosing of epinephrine, norepinephrine or isoproterenol is not possible due to rapid metabolism of the catechol nucleus in gastrointestinal mucosa and liver.  Therefore, these agents must be given by routes that avoid the stomach.

 

 

Epinephrine

 

Epinephrine is a very versatile drug that has many uses and is administered in many dosage forms. 

 

Routes of administration and uses of Epinephrine

 

1) Epinephrine can be given by injection (s.c., i.m. i.v) or inhalation for the treatment of respiratory distress or bronchspasm caused for example by asthma (i.e. status asthmaticus) or anaphylaxis as a result of allergic responses.  A particular note is made of the Epipen that can be carried by individuals prone to bronchospasm.  In this instance the salutatory effects of epinephrine would be its bronchodilatory actions at the beta2-receptor.

 

 

2) Epinephrine is also used in cardiopulmonary resuscitation because of its ability to activate the myocardial beta1-receptor. 

 

3) Epinephrine can be given by injection or topically in combination with local anesthetics  such as articaine, bupivacaine or lidocaine to prolong the duration of anesthetic action.  This combination is used because epinephrine can induce vasoconstriction thus limiting the diffusion of the local anesthetic from the site of injection.  This not only prolongs the duration of actions of the local anesthetic action but also reduces the toxicity of the local anesthetic by limiting its systemic absorption.  For example, lidocaine in toxic doses can produce cardiac arrthythmias and convulsions.

 

4) Epinephrine can also be topically applied in surgical procedures to induce vasoconstriction and thus reduce blood loss.               

                                               

5) Clinical studies have shown that epinephrine blood levels increase following its intraoral administration.  The risk of this increase is dependent on characteristics of the patient. For example, hypertensive patients or those with other cardiovascular disease or patients taking other drugs that affect sympathetic nervous system function are at higher risk than patients without these conditions.  Systemically absorbed epinephrine could also increase heart rate and exacerbate cardiac rhythm disturbances or myocardial ischemia                              

 

 

 

Norepinephrine and Isoproterenol

 

For the same reasons as epinephrine, isoproterenol can be used to treat bronchospasm Norepinephrine can be used to produce vasoconstriction, via the alpha1-receptor,  in the treatment of cardiogenic or septic shock.