Differential Regulation of Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase Isozymes

Summary

Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions that occur between the cyclic nucleotide and Ca²⁺ second messenger systems. This enzyme is characterized by a high Km for cAMP and a lower Km for cGMP. CaMPDE exists in different isozymic forms that exhibit distinct molecular and /or catalytic properties. Four CaMPDE isozymes have been purified close to homogeneity from bovine tissue in this laboratory. Immunologic, kinetic, and regulatory characterization have revealed subtle differences among these enzymes. The 63 kD CaMPDE is kinetically distinct from the other three isozymes. This isozyme is also further distinguished from other isozymes by the ginsenoside inhibition study. Ginsenosides are found to be potent inhibitors of brain 60 kD, heart and lung CaMPDE isozymes, but not of the brain 63 kD isozyme. The three other members — brain 60 kD CaMPDE, heart CaMPDE, and lung CaMPDE — show differential activation by calmodulin and Ca²⁺. These observations are consistent with the notion that differential regulation by calmodulin and Ca²⁺ kinetics inhibition are important functions of these isozymes that provide fine-tuning mechanisms for calmodulin action.

Kakiuchi and Yamazaki [1] originally demonstrated the existence of a Ca²⁺-stimulated cyclic nucleotide phosphodiesterase in rat brain. In addition, they discovered an endogenous brain protein factor that could enhance the Ca²⁺ sensitivity of the enzyme [2]. It was subsequently established [3] that the protein factor was identical to the protein activator of cyclic nucleotide phosphodiesterase (later called calmodulin), which was originally discovered by Cheung [4,5], and that stimulation of the cyclic nucleotide phosphodiesterase required the simultaneous presence of both Ca²⁺ and calmodulin (CaM) [3,6]. The activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) was found to be widely distributed in mammalian tissues and other eukaryotes [7–11]. CaMPDE is one of the most intensively studied and best characterized of the multiple phosphodiesterases (PDEs). The enzyme has been purified to homogeneity and characterized in terms of molecular properties [12-19]. Initially, it was thought that a single form of enzyme existed in all tissues [8], but with the availability of specific monoclonal antibodies it has become clear that the CaMPDE exists as tissue-specific and immunologic distinct isozymes [18–24].

The CaMPDE isozymes show subtle differences in kinetic and regulatory properties and shed new light on their functions. They are differentially regulated by CaM [19,25,26], Ca²⁺ [27] and/or by protein phosphorylation mechanisms [19,28-30]. The purpose of this chapter is to summarize some of the significant advances that have been carried in our laboratory.