Which of the following best predicts the effect of inhibiting cyclic AMP AMP phosphodiesterase in a muscle cell stimulated by epinephrine?

Intracellular concentrations of cAMP play an important second messenger role in regulating cardiac muscle contraction. Activation of the sympathetic nervous system releases the neurotransmitter norepinephrine and increases circulating catecholamines (epinephrine and norepinephrine). These catecholamines bind primarily to beta1-adrenoceptors in the heart that are coupled to Gs-proteins. This activates adenylyl cyclase to form cAMP from ATP. Increased cAMP, through its coupling with other intracellular messengers, increases contractility (inotropy), heart rate (chronotropy) and conduction velocity (dromotropy). Cyclic-AMP is broken down by an enzyme called cAMP-dependent phosphodiesterase (PDE). The isoform of this enzyme that is targeted by currently used clinical drugs is the type 3 form (PDE3). Inhibition of this enzyme prevents cAMP breakdown and thereby increases its intracellular concentration. This increases cardiac inotropy, chronotropy and dromotropy. PDE3 inhibitors can be thought of as a backdoor approach to cardiac stimulation, whereas β-agonists go through the front door to produce the same cardiac effects.

Blood vessels

Cardiovascular Actions of cAMP-dependent PDE (type3) Inhibitors

Systemic Circulation

• Vasodilation
• Increased organ perfusion
• Decreased systemic vascular resistance
• Decreased arterial pressure

Cardiopulmonary

• Increased contractility and heart rate
• Increased stroke volume and ejection fraction
• Decreased ventricular preload
  (secondary to increased output)
• Decreased pulmonary capillary wedge pressure

The cardiac and vascular effects of cAMP-dependent PDE inhibitors cause cardiac stimulation, which increases cardiac output, and reduced systemic vascular resistance, which tends to lower arterial pressure. Because cardiac output increases and systemic vascular resistance decreases, the change in arterial pressure depends on the relative effects of the PDE inhibitor on the heart versus the vasculature. At normal therapeutic doses, PDE3 inhibitors such as milrinone have a greater vascular than cardiac effect so that arterial pressure is lowered in the presence of augmented cardiac output. Because of the dual cardiac and vascular effects of these compounds, they are sometimes referred to as "inodilators."

Other actions

PDE3 inhibitors also decrease platelet aggregation by increasing platelet cAMP. However, only cilostazol (see below) is used for this purpose in the treatment of intermittant claudication (ischemic leg pain associated with leg movement).

General Pharmacology of cGMP-Dependent Phosphodiesterase Inhibitors (PDE5)

There is a second isoenyme form of PDE in vascular smooth muscle that is a cGMP-dependent phosphodiesterase. The type 5 isoform of this enzyme (PDE5) is found in the corpus cavernosum of the penis and in vascular smooth muscle. This enzyme is responsible for breaking down cGMP that forms in response to increased nitric oxide (NO). Increased intracellular cGMP inhibits calcium entry into the cell, thereby decreasing intracellular calcium concentrations and causing smooth muscle relaxation (click here for details).

NO also activates K+ channels, which leads to hyperpolarization and relaxation. Finally, NO acting through cGMP can stimulate a cGMP-dependent protein kinase that activates myosin light chain phosphatase, the enzyme that dephosphorylates myosin light chains, which leads to relaxation. Therefore, inhibitors cGMP-dependent phosphodiesterase, by increasing intracellular cGMP, enhance smooth muscle relaxation and vasodilation, and cause penile erection.

Therapeutic Indications

The cardiostimulatory and vasodilatory actions of PDE3 inhibitors make them suitable for the treatment of heart failure. Arterial dilation reduces afterload on the failing ventricle and leads to an increase in stroke volume and ejection fraction, as well as increases organ perfusion. Reducing the afterload leads to a secondary decrease in preload on the heart that helps to improve the mechanical efficiency of dilated hearts and to reduce ventricular wall stress and the oxygen demands placed on the failing heart. The cardiostimulatory effects of these drugs increase inotropy, which further enhances stroke volume and ejection fraction. Tachycardia, however, also results, and this is not beneficial; therefore, doses are used that minimize the positive chronotropic actions of the drug. A baroreceptor reflex, which occurs in response to hypotension, may contribute to the tachycardia. Clinical trials have shown that long-term therapy with PDE3 inhibitors increases mortality in heart failure patients; therefore, these drugs are not used for long-term, chronic therapy. They are very useful, however, in treating acute, decompensated heart failure or temporary bouts of decompensated chronic failure. They are not used as a monotherapy. Instead, they are used in conjunction with other treatment modalities such as diuretics, ACE inhibitors, beta-blockers or digitalis.

The somewhat selective vasodilatory actions of PDE5 inhibitors have made these compounds very useful in the treatment of male erectile dysfunction. The PDE5 inhibitor sildenafil is also approved for the treatment of pulmonary hypertension.

Several different PDE inhibitors are available for clinical use:

• PDE3 inhibitors
  • milrinone
  • inamrinone (formerly amrinone)
  • cilostazol
• PDE5 inhibitors

The PDE3 inhibitors (except cilostazol) are used for treating acute, decompensated heart failure, whereas the PDE5 inhibitors are used for treating male erectile dysfunction and pulmonary hypertension. Note that the PDE3 inhibitors used in acute heart failure end in "one," whereas the PDE5 inhibitors end in "fil".

Inhibition of platelet aggregation, along with vasodilation, is an important mechanism of action for cilostazol, which is used in the treatment of intermittant claudication in peripheral arterial disease. Cilostazol appears to have less cardiostimulatory effects than milrinone.

Side Effects and Contraindications

PDE3 inhibitors

Milrinone and inamrinone are not used in the treatment of chronic heart failure because clinical trials have shown that long-term use of these drugs worsen outcome. The most common and severe side effect of PDE3 inhibitors is ventricular arrhythmias in about 12% of patients, some of which may be life-threatening. Headaches and hypotension occur in about 3% of patients. These side effects are not uncommon for drugs that increase cAMP in cardiac and vascular tissues, other examples being β-agonists.

PDE5 inhibitors

The most common side effects for PDE5 inhibitors include headache and cutaneous flushing, both of which are related to vascular dilation caused by increased vascular cGMP. There is clinical evidence that nitrodilators may interact adversely with PDE5 inhibitors. The reason for this adverse reaction is that nitrodilators stimulate cGMP production while PDE5 inhibitors inhibit cGMP degradation. When combined, these two drug classes greatly potentiate cGMP levels, which can lead to hypotension and impaired coronary perfusion.

Revised 01/23/21

1. The proliferation of thyroid cells is stimulated by hormones that activate a receptor coupled to Gs. How would inhibitors of cAMP phosphodiesterase affect the proliferation of these cells?

Inhibition of cAMP phosphodiesterase would result in elevated levels of cAMP, which would stimulate cell proliferation.

2. The epinephrine receptor is coupled to Gs, whereas the acetylcholine receptor (on heart muscle cells) is coupled to Gi. Suppose you were to construct a recombinant molecule containing the extracellular sequences of the epinephrine receptor joined to the cytosolic sequences of the acetylcholine receptor. What effect would epinephrine have on cAMP levels in cells expressing such a recombinant receptor? What would be the effect of acetylcholine?

The recombinant molecule would function as an epinephrine receptor coupled to Gi. Epinephrine would therefore inhibit adenylyl cyclase, lowering intracellular cAMP levels. Acetylcholine would have no effect, since it would not bind to the recombinant receptor.

3. Platelet-derived growth factor (PDGF) is a dimer of two polypeptide chains. What would be the predicted effect of PDGF monomers on signaling from the PDGF receptor?

PDGF monomers would not induce receptor dimerization. Since this is the first critical step in signaling from receptor protein-tyrosine kinases, they would be unable to stimulate the PDGF receptor.

4. How would overexpression of protein phosphatase 1 affect the induction of cAMP-inducible genes in response to hormone stimulation of appropriate target cells? Would protein phosphatase 1 affect the function of cAMP-gated ion channels involved in odorant reception?

Protein phosphatase 1 dephosphorylates serine residues that are phosphorylated by protein kinase A. Cyclic AMP-inducible genes are activated by CREB, which is phosphorylated by protein kinase A, so overexpression of protein phosphatase 1 would inhibit their induction. However, protein phosphatase 1 would not affect the activity of cAMP-gated ligand channels, since these channels are opened directly by cAMP binding rather than by protein phosphorylation.

5. Protein kinase C-α (PKC-α) and protein kinase C-ε (PKC-ε) are two different members of the protein kinase C family, which differ in their regulation. PKC-α requires both Ca2+ and diacylglycerol for activation, whereas PKC-ε requires only diacylglycerol. How would hydrolysis of the phospholipids PIP2 and phosphatidylcholine by phospholipase C affect the activities of these different PKC family members?

Hydrolysis of PIP2 by phospholipase C yields both diacylglycerol and IP3, which signals the release of Ca2+ from the endoplasmic reticulum. PIP2 hydrolysis can therefore activate both PKC-α and PKC-ε. Hydrolysis of phosphatidylcholine yields diacylglycerol but not IP3; consequently, phosphatidylcholine hydrolysis is sufficient to activate PKC-ε but not PKC-α.

6. Dominant negative mutants of both Ras and Raf block growth factor-stimulated cell proliferation. The inhibitory effects of dominant negative Ras are overcome by expression of activated Raf. Would you expect activated Ras similarly to overcome the inhibitory effects of dominant negative Raf? How about activated MEK?

Raf acts downstream of Ras in the MAP kinase pathway. Activated Raf can therefore bypass the effects of dominant negative Ras, but activated Ras cannot overcome the inhibitory effects of dominant negative Raf. MEK acts downstream of Raf, so activated MEK can overcome the effects of dominant negative Ras or Raf.