cGMP-dependent protein kinases (cGK) are serine/threonine kinases that are widely distributed in eukaryotes. Two genes--prkg1 and prkg2--code for cGKs, namely cGKI and cGKII. In mammals, two isozymes, cGKIalpha and cGKIbeta, are generated from the prkg1 gene. The cGKI isozymes are prominent in all types of smooth muscle, platelets, and specific neuronal areas such as cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The cGKII prevails in the secretory epithelium of the small intestine, the juxta-glomerular cells, the adrenal cortex, the chondrocytes, and in the nucleus suprachiasmaticus. Both cGKs are major downstream effectors of many, but not all signalling events of the NO/cGMP and the ANP/cGMP pathways. cGKI relaxes smooth muscle tone and prevents platelet aggregation, whereas cGKII inhibits renin secretion, chloride/water secretion in the small intestine, the resetting of the clock during early night, and endochondreal bone growth. cGKs are also modulators of cell growth and many other functions.

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKIdependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP 3 RI)-associated protein (IRAG), which decreases hormone-induced IP 3 -dependent Ca 2 þ release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP 3 RI interaction and resulted in hypomorphic IRAG D12/D12 mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol-and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG D12/D12 mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca 2 þ ] i were not decreased by cGMP in aortic smooth muscle cells from IRAG D12/D12 mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG D12/D12 mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP 3 RI.

Abstract-Smooth muscle expresses the I␣ and the I␤ isoforms of cGMP-dependent protein kinase I (cGKI). Inactivation of the murine cGKI gene prkg1 leads to multiple phenotypes and premature death at Ϸ6 weeks. We reconstituted mice with the cGKI␣ or -I␤ isozyme to test which isozyme was needed to support basic smooth muscle functions. Mice were generated by gene targeting. The cGKI␣ or the -I␤ coding sequences were placed under the control of the SM22␣ promoter to express either isoform selectively in smooth muscle cells (SM-I␣ or SM-I␤ transgene). To generate smooth muscle-specific cGKI␣ or cGKI␤ rescue mice, the SM-I␣ or SM-I␤ transgenes were crossed on a cGKI Ϫ/Ϫ genetic background. The levels of cGKI␣ or -I␤ expression were comparable to endogenous cGKI expression in wild-type aortic and intestinal smooth muscles. In cGKI␣ or -I␤ rescue mice, expression of the isozymes was not detectable in non-smooth muscle tissues and cells. Median survival time of the I␣ and I␤ rescue mice was 52 weeks. Both isozymes mediated the 8-bromo-cGMP-induced relaxation of precontracted jejunum and aorta muscle strips. Activation of both isozymes reduced hormone-or K ϩ -induced [Ca 2ϩ ] i levels. The cGKI␣ and cGKI␤ rescue mice did not show a significant difference in intestinal passage time of BaSO 4 in comparison with wild-type animals. Telemetric blood pressure measurements in conscious freely moving animals did not show differences between rescues and control mice in basal blood pressure and its regulation by DETA-NO, sodium nitroprusside, carbachol, or Y-27632. These results show that cGKI in smooth muscle is essential and that either cGKI isozyme alone can rescue basic vascular and intestinal smooth muscle functions. Key Words: cGMP kinase isozymes Ⅲ PKG Ⅲ nitric oxide Ⅲ smooth muscle Ⅲ blood pressure T he NO/cGMP signaling cascade plays an essential role in vascular smooth muscle (SM) relaxation, and clinical studies indicate that endothelium-derived NO is involved in normal and pathological blood pressure regulation in humans. [1][2][3] The important effector of cGMP, cGMP-dependent protein kinase I (cGKI), is highly expressed in SM. 4 Conventional deletion of the gene for cGKI in mice leads to multiple phenotypes, including severe gastrointestinal disturbances and elevated blood pressure, leading to premature death of the animals. 5 The cGKI gene generates 2 isoforms, cGKI␣ and cGKI␤, that differ only in their individual N termini (the first 90 to 100 residues), which are encoded by 2 alternatively used exons. 6,7 Both isoforms are expressed together in various SMs. 8,9 Strong evidence has been published that these isozymes interact with different proteins and affect SM relaxation through different mechanisms. 10 -13 cGKI␣ interacts specifically with MYPT1 (myosin-interacting subunit of myosin phosphatase 1) 12 and with RGS-2 (regulator of G protein signaling 2), 13 whereas cGKI␤ shows specificity for inositol 1Ј,4Ј,5Ј-triphosphate receptor-associated G kinase substrate (IRAG). 10,11 However, in vitro data from Feil et al...

Long-term potentiation (LTP) of inputs relaying sensory information from cortical and thalamic neurons to principal neurons in the lateral amygdala (LA) is thought to serve as a cellular mechanism for associative fear learning. Nitric oxide (NO), a messenger molecule widely implicated in synaptic plasticity and behavior, has been shown to enhance LTP in the LA as well as consolidation of associative fear memory. Additional evidence suggests that NO-induced enhancement of LTP and amygdala-dependent learning requires signaling through soluble guanylyl cyclase (sGC) and cGMP-dependent protein kinase (cGK). Mammals possess two genes for cGK: the prkg1 gene gives rise to the cGK type I isoforms, cGKI␣ and cGKI␤, and the prkg2 gene encodes the cGK type II. Reportedly, both cGKI and cGKII are expressed in the amygdala, and cGKII is involved in controlling anxiety-like behavior. Because selective pharmacological tools for individual cGK isoforms are lacking, we used different knock-out mouse models to examine the function of cGKI and cGKII for LTP in the LA and pavlovian fear conditioning. We found robust expression of the cGKI specifically in the LA with cGKI␤ as the prevailing isoform. We further show a marked reduction of LTP at both thalamic and cortical inputs to the LA and a selective impairment of auditory-cued fear memory in cGKI-deficient mutants. In contrast, cGKII null mutants lack these phenotypes. Our data suggest a function of cGKI, likely the ␤ isoform, in the LA, supporting synaptic plasticity and consolidation of fear memory.