The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse trnscriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMPdependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this done, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKH isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK H loalization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK H poly(A) RNA form was 4.8 kb, with minor forms of6.2 and 3.1 kb. The cGK H RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.

IntroductionCertain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea.

AbstractcGMP-based regulatory systems are vital for counteracting the renin-angiotensin system (RAS) which promotes volume expansion and high blood pressure. Natriuretic peptides and nitric oxide acting through their second messenger cGMP normally increase natriuresis and diuresis, and regulate renin release; however, the severe pathological state of cardiac heart failure is characterized by elevated levels of atrial natriuretic peptide that are no longer able to effectively oppose exaggerated RAS effects. There is presently limited information on the intracellular effectors of cGMP actions in the kidney. Recently we reported the cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), which is highly enriched in intestinal mucosa but was also detected for the first time in kidney. In the present study, cGK II was localized to juxtaglomerular (JG) cells, the ascending thin limb (ATL), and to a lesser extent the brush border of proximal tubules. An activator of renin gene expression, the angiotensin II type I receptor inhibitor, losartan, increased cGK II mRNA and protein three to fourfold in JG cells. In other experiments, water deprivation increased cGK II mRNA and protein three to fourfold in the inner medulla where both cGK II, and a kidney specific Cl

The discovery that collateral development after progressive coronary stenosis proceeds by means of DNA synthesis, mitosis and proliferation of endothelial and smooth muscle cells in preformed small interconnecting arterioles (canine heart) and capillaries (porcine heart) has stimulated research into the molecular mechanisms of vascular growth. Growth is tightly controlled under physiologic conditions, and several factors must act in concert to overcome control. Because the result of growth is a much larger orderly structure of complex design, we expect the existence of a genetic blueprint for its construction. Peptide growth factors have recently been isolated from a variety of organs, including the heart. We have provided experimental evidence that the heparin-binding growth factor beta-ECGF shows an increased transcription in growing pig collateral vessels. Because the chain of events probably originates in the ischemic cardiac myocyte, it appears logical to search there for the initiating factor. In addition to local production, growth factors can also be transported into ischemic myocardium by blood-borne cells. Monocytes adhere to altered endothelium in a potentially ischemic region and start to produce growth factors in situ. Platelets are rich sources of transforming growth factor-beta (TGF-beta), platelet-derived endothelial cell growth factor (PDECGF) and platelet-derived growth factor (PDGF), all of which are known angiogenic factors or mitogens.

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