Naphthalenesulfonamides such as N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7) are potent calmodulin (CaM) antagonists and act upon several protein kinases at higher concentration. When the naphthalene ring was replaced by isoquinoline, the derivatives were no longer CaM antagonists but retained the ability to inhibit protein kinases, and some of the derivatives exhibited selective inhibition toward a certain protein kinase. cAMP-dependent, cGMP-dependent, and Ca2+-phospholipid-dependent (protein kinase C) protein kinases were inhibited significantly by addition of 10(-6) M N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (H-8) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). H-8 was the most active of the inhibitors in this series and inhibited more markedly cyclic nucleotide dependent protein kinases, than other kinases, while the derivative with the sulfonylpiperazine residue (H-7) was the most potent in inhibiting protein kinase C. Apparent Ki values of H-8 were 0.48 and 1.2 microM for cGMP-dependent and cAMP-dependent protein kinases, respectively, and the Ki value of H-7 for protein kinase C was 6 microM. Both the holoenzyme and the catalytic subunit (or fragment), which is active without an enzyme activator, are susceptible to these compounds with a similar concentration dependency, thereby indicating that the inhibitory effect is attributed to the direct interaction of the compound with the active center of the enzyme but not with the enzyme activator. The inhibitions were freely reversible and of the competitive type with respect to ATP and of the noncompetitive type with respect to the phosphate acceptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C.

The role of calcium ions (Ca2+) in cell function is beginning to be unraveled at the molecular level as a result of recent research on calcium-binding proteins and particularly on calmodulin. These proteins interact reversibly with Ca2+ to form a protein . Ca2+ complex, whose activity is regulated by the cellular flux of Ca2+. Many of the effects of Ca2+ appear to be exerted through calmodulin-regulated enzymes.

Calmodulin plays a pivotal role in cellular regulation

https://www.jbc.org/content/247/4/1106.full.pdf

Radioimmunoassay for Cyclic Nucleotides I. PREPARATION OF ANTIBODIES AND IODINATED CYCLIC NUCLEOTIDES

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.

https://www.pnas.org/content/pnas/88/7/2811.full.pdf

Neuronal NADPH diaphorase is a nitric oxide synthase.

Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells.

Adenosine 3′,5′-monophosphate-dependent protein kinase activity was found in about thirty sources including many mammalian tissues as well as species representative of eight different invertebrate phyla. The data support a unifying theory for the mechanism of action of adenosine 3′,5′-monophosphate, namely that its many and diverse effects are mediated through activation of tissue-specific protein kinases.

https://www.pnas.org/content/pnas/64/4/1349.full.pdf

Cyclic nucleotide-dependent protein kinases, iv. widespread occurrence of adenosine 3′,5′-monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom

Cyclic Nucleotide-dependent Protein Kinases III. PURIFICATION AND PROPERTIES OF ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE FROM BOVINE BRAIN

Cyclic nucleotide-dependent protein kinases. VI. Isolation and partial purification of a protein kinase activated by guanosine 3',5'-monophosphate.

Effects of acetylcholine and of agents that mimic or block its physiological actions have been studied upon concentrations of guanosine 3′:5′-cyclic monophosphate (cyclic GMP) and adenosine 3′:5′-cyclic monophosphate (cyclic AMP) in slices of mammalian cerebral cortex, heart ventricle, and ileum. Acetylcholine, and cholinomimetic agents with a predominantly muscarinic action, such as methacholine, bethanechol, and pilocarpine, induced an increase in the concentration of cyclic GMP, accompanied by no change or a slight decrease in the concentration of cyclic AMP, in all three tissues studied. Tetramethylammonium, a cholinomimetic agent with a predominantly nicotinic action, on the other hand, did not significantly alter concentrations of either cyclic GMP or cyclic AMP. The increase in the tissue content of cyclic GMP induced by acetylcholine and its muscarinic analogs was antagonized by atropine, a muscarinic blocking agent, but not by hexamethonium, a nicotinic blocking agent. 

These and other results suggest the generalization that the interaction of acetylcholine with muscarinic receptors, but not with nicotinic receptors, causes an increase in the concentration of cyclic GMP. The data are compatible with the hypothesis that cyclic GMP may mediate the muscarinic actions of acetylcholine. 

The antagonistic actions of cholinergic and adrenergic agents on the physiology of contraction of intestinal smooth muscle and of cardiac muscle are reflected in, and may result from, antagonistic actions of these compounds on the levels of both cyclic GMP and cyclic AMP in these tissues.

https://www.pnas.org/content/pnas/69/11/3287.full.pdf

Role of Muscarinic Cholinergic Receptors in Regulation of Guanosine 3′:5′-Cyclic Monophosphate Content in Mammalian Brain, Heart Muscle, and Intestinal Smooth Muscle

J.F. Kuo

Papers

Cyclic nucleotide-dependent protein kinases, iv. widespread occurrence of adenosine 3′,5′-monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom

Cyclic Nucleotide-dependent Protein Kinases III. PURIFICATION AND PROPERTIES OF ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE FROM BOVINE BRAIN

Cyclic nucleotide-dependent protein kinases. VI. Isolation and partial purification of a protein kinase activated by guanosine 3',5'-monophosphate.

Role of Muscarinic Cholinergic Receptors in Regulation of Guanosine 3′:5′-Cyclic Monophosphate Content in Mammalian Brain, Heart Muscle, and Intestinal Smooth Muscle

An Adenosine 3',5'-Monophosphate-dependent Protein Kinase from Escherichia coli